CN106795519A - Method for generating glutaric acid and glutaric acid methyl esters - Google Patents

Method for generating glutaric acid and glutaric acid methyl esters Download PDF

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CN106795519A
CN106795519A CN201580038453.6A CN201580038453A CN106795519A CN 106795519 A CN106795519 A CN 106795519A CN 201580038453 A CN201580038453 A CN 201580038453A CN 106795519 A CN106795519 A CN 106795519A
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A.L.博特斯
A.V.E.康拉迪
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Invista Textiles UK Ltd
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Technology Of English Weida LLC
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    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/99—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with other acceptors (1.2.99)
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    • C12Y301/01085—Pimelyl-[acyl-carrier protein] methyl ester esterase (3.1.1.85)

Abstract

Presents is described for generating glutaric acid, 5 aminovaleric acids, 5 hydroxypentanoic acids, cadaverine or 1; the bio-chemical pathway of 5 pentanediols; it is carried out by forming one or two functional end-group being made up of carboxyl, amine or oh group in C5 main chain substrates, such as malonyl CoA or malonyl [acp].

Description

Method for generating glutaric acid and glutaric acid methyl esters
Cross-reference to related applications
This application claims the U.S. Provisional Application No. 62/012,722 submitted on June 16th, 2014 and in June, 2014 The rights and interests of the U.S. Provisional Application No. 62/012,586 submitted to for 16th, the disclosure of which is incorporated herein by reference in their entirety.
Invention field
The present invention relates to improve the polypeptide with carboxylate reductase activity to the active method of dicarboxylic acids, its by using Dicarboxylic acids enzymatic is converted into methyl esters and carried out by the polypeptide with malonyl-CoA methyl transferase activities.The invention further relates to There is malonyl-[acp] O- transmethylases, esterase, dehydratase, hydrase, dehydrogenase, thioesters in using one or more The polypeptide biosynthesis glutaric acid of enzyme, reversible CoA ligase, CoA- transferases, carboxylate reductase, or ω-transaminase activity, The method of 5- aminovaleric acids, cadaverine, 5- hydroxypentanoic acids, or 1,5-PD (hereinafter " C5 building blocks "), and generate such C5 The recombinant host of building block.
Background technology
Nylon is polyamide, and it is typically by diamines and the condensation polymerization (condensation of dicarboxylic acids Polymerization) synthesize.It is likewise possible to pass through the condensation polymerization generation nylon of lactams.A kind of Buddhist nun of generally existing Dragon is nylon 6,6, its condensation polymerization generation for passing through hexamethylene diamine (HMD) and adipic acid.Can be by caprolactam Ring-opening polymerisation generation nylon 6 (Anton＆Baird, Polyamides Fibers, Encyclopedia of Polymer Science and Technology,2001)。
Nylon 5, nylon 5,5 and other variants including C5 monomers represent in numerous applications with nylon 6 and the phase of nylon 6,6 Than the novel polyamide with breeding property.Nylon 5 is generated by the polymerization of 5- aminovaleric acids, and passes through glutaric acid and cadaverine Condensation polymerization generates nylon 5,5.In the absence of economically viable petrochemistry route producing the monomer of nylon 5 and nylon 5,5.
In view of without economically feasible petrochemistry raw material monomer, biotechnology provides alternative side by living things catalysis Method.Living things catalysis is to use biocatalyst, and such as enzyme carries out the biochemical transformation of organic compound.
The raw material and petrochemical feedstocks of biogenetic derivation are both used for the feasible parent material of living things catalysis process.
Thus, for this background, it is clear that need for producing glutaric acid, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine With the continuable method of one or more in 1,5-PD (hereinafter referred to as " C5 building blocks "), wherein methods described is base In living things catalysis.
However, the prokaryotes or eucaryote of wild type do not generate C5 building blocks to extracellular environment excessively.But, penta Diacid, the metabolism of 5- aminovaleric acids and cadaverine has been reported.
Many bacteriums and yeast are via beta oxidation using dicarboxylic acids adipic acid as metabolite centered on carbon source Efficient Conversion. The decarboxylation of the glutaric acid of coacetylase (CoA) activation to crotonyl-CoA promotes via the further catabolism of beta oxidation.
To anaerobic bacteria, such as Clostridium viride report metabolism (the Buckel et of 5- aminovaleric acids al.,2004,Arch.Microbiol.,162,387-394).It is likewise possible to cadaverine is degraded into acetic acid and butyric acid (Roeder and Schink,2009,Appl.Environ.Microbiol.,75(14),4821–4828)。
The principle of optimization is described, and microorganism adjusts their biochemistry network to support maximum biomass (biomass) Growth.Beyond the need for expressing heterologous approach in host organisms, Carbon flux is directed to the C5 construction units for serving as carbon source Rather than biomass growth component and principle of optimization contradiction.For example, by n-butyl alcohol approach from fusobacterium (Clostridium) species Be transferred to other production bacterial strains with the production performance of natural producer compared with often differ an order of magnitude (Shen et al., Appl.Environ.Microbiol.,2011,77(9):2905–2915)。
Being effectively synthesized for 5 carbocyclic aliphatic race backbone precursors is that functional end-group, such as carboxyl, amine are formed in C5 aliphatic backbones Or synthesize the crucial consideration of one or more C5 building blocks before oh group.
Summary of the invention
Presents is at least partially based on following discoveries：Can build for from malonyl-[acp] or malonyl-CoA The bio-chemical pathway of 5 carbon backbone precursors, such as glutaryl-[acp], glutaryl-CoA or glutaric acid methyl esters is generated, One or two functional group, i.e. carboxyl, amine or hydroxyl can be wherein formed, causes following one or more of synthesis：Glutaric acid, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine (being also called 1,5 pentanediamines), and 1,5-PD (hereinafter " C5 building blocks ").Penta Diacid semialdehyde (being also called 5- oxopentanoic acids) can be generated as the intermediate of other products.Glutaric acid and glutarate, 5- hydroxyls Base valeric acid and 5- hydroxypentanoic acids salt, 5- oxopentanoic acids and 5- oxopentanoic acids salt and 5- aminovaleric acids and 5- aminovaleric acids salt can be mutual Change and use, refer to the compound of any its neutral or ionized form, including its any salt form.It will be appreciated by those skilled in the art that Specific form will be depending on pH.
In some embodiments, can via conversion to glutaryl-[acp] methyl esters or glutaryl-CoA methyl esters, Then glutaryl-[acp] methyl esters or the de- esterification (de-esterification) of glutaryl-CoA methyl esters are respectively by (i) Glutaryl-[acp] or glutaryl-CoA, or (ii) hydrolyzes glutaryl-[acp] methyl esters or glutaryl-CoA methyl esters It is glutaric acid methyl esters, the C5 aliphatic masters for being converted into C5 building blocks is formed from malonyl-[acp] or malonyl-CoA Chain.Referring to Fig. 1-3.
In some embodiments, the enzyme in the approach of C5 aliphatic backbones is produced purposefully to be walked containing irreversible enzyme Suddenly.
In some embodiments, it is possible to use esterase, thioesterase, reversible CoA ligase, CoA transferases, aldehyde dehydrogenation Enzyme, 7- oxo-heptanoic acids dehydrogenase, 6- oxo caproic acid dehydrogenases or 5- oxopentanoic acid dehydrogenases enzymatic form terminal carboxyl groups.Ginseng See Fig. 4.
In some embodiments, it is possible to use ω-transaminase or deacetylase enzymatic form terminal amine group.Referring to Fig. 5-7.
In some embodiments, it is possible to use alcohol dehydrogenase, 4 hydroxybutyric acid dehydrogenase, 5- hydroxypentanoic acids dehydrogenase and 6 hydroxycaproic acid dehydrogenase enzymatic forms terminal hydroxyl group.Referring to Fig. 8 and Fig. 9.
Thioesterase can be with SEQ ID NO.22-23, SEQ ID NO:The amino acid sequence listed in 17-18 has at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).
ω-transaminase can have at least 70% sequence with any one of amino acid sequence listed in SEQ ID NO.8-13 Row homogeneity (homology) (for example, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).
Carboxylate reductase (for example being combined with Phosphopantetheinyl transferase) can form end in product is formed Terminal aldehyde group group is used as intermediate.Carboxylate reductase can have with any one of amino acid sequence listed in SEQ ID NO.2-7 Have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, Or 100%).
In an aspect, the method that presents is characterised by the biosynthesis glutaric acid semialdehyde methyl esters in recombinant host. Method includes thering is thioesters using at least one polypeptide with malonyl-CoA O- methyl transferase activities and at least one At least one enzymatic in malonyl-[acp] and malonyl-CoA is converted into glutaric acid methyl esters by the polypeptide of enzymatic activity.
In some embodiments, at least one polypeptide with malonyl-CoA O- methyl transferase activities is used Malonyl-[acp] enzymatic is converted into malonyl-[acp] methyl esters.Can be using at least one with the work being selected from the group Malonyl-[acp] methyl esters enzymatic is converted into glutaryl-[acp] methyl esters by the polypeptide of property：Synthase activity, dehydrogenase activity, Dehydratase activity, and reductase activity.At least one polypeptide with thioesterase activity can be used by glutaryl-[acp] Methyl esters enzymatic is converted into glutaric acid methyl esters.
In some embodiments, at least one polypeptide with malonyl-CoA O- methyl transferase activities is used Malonyl-CoA enzymatics are converted into malonyl-CoA methyl esters.
In some embodiments, using it is at least one with the active polypeptide being selected from the group by malonyl-CoA first Esterase promotees to be converted into glutaryl-CoA methyl esters：Synthase activity, beta-Ketothiolase activity, dehydrogenase activity, hydratase activity, and Reductase activity.The method of claim 7, wherein using at least one polypeptide with thioesterase activity by glutaryl-CoA Methyl esters enzymatic is converted into glutaric acid methyl esters.
Polypeptide with malonyl-CoA O- methyl transferase activities can be with SEQ ID NO:The amino listed in 21 Acid sequence have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with reductase activity can be with SEQ ID NO:The amino acid sequence listed in 19 or 20 has at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
In some embodiments, method further includes there is carboxylate reductase activity using at least one in host Polypeptide glutaric acid methyl esters enzymatic is converted into glutaric acid semialdehyde methyl esters.Can increase with phosphopantetheine transferase The polypeptides in combination of strong agent activity uses the polypeptide with carboxylate reductase activity.
In some embodiments, method further includes to be incited somebody to action with the active polypeptide being selected from the group using at least one Glutaric acid semialdehyde methyl esters enzymatic is converted into 5- aminovaleric acids：ω-transaminase activity and esterase active.Method may further include 5- aminovaleric acid enzymatics are converted into cadaverine with the active polypeptide being selected from the group using at least one：Carboxylate reductase is lived Property, ω-transaminase activity, N- acetyl transferase activity, alcohol dehydrogenase activity, and deacetylase activity.Can with have The polypeptides in combination of phosphopantetheine transferase reinforcing agent activity uses the polypeptide with carboxylate reductase activity.
In some embodiments, method further includes to be incited somebody to action with the active polypeptide being selected from the group using at least one Glutaric acid methyl esters enzymatic is converted into 5- oxopentanoic acids：Carboxylate reductase activity and esterase active.Method may further include to be made 5- oxopentanoic acid enzymatics are converted into cadaverine with the active polypeptide being selected from the group with least one：Carboxylate reductase activity, With ω-transaminase activity activity.Can be used with the polypeptides in combination with phosphopantetheine transferase reinforcing agent activity Polypeptide with carboxylate reductase activity.
In some embodiments, method further includes to use at least one polypeptide with esterase active by glutaric acid Semialdehyde methyl esters enzymatic is converted into 5- hydroxypentanoic acids.Method may further include using at least one with many of dehydrogenase activity Glutaric acid semialdehyde methyl esters enzymatic is converted into 5- hydroxypentanoic acids by peptide.In some embodiments, method may further include makes 5- hydroxypentanoic acid enzymatics are converted into cadaverine with the active polypeptide being selected from the group with least one：Carboxylate reductase activity, ω-transaminase activity, and alcohol dehydrogenase activity.Can be with the polypeptide with phosphopantetheine transferase reinforcing agent activity It is applied in combination the polypeptide with carboxylate reductase activity.In some embodiments, method may further include using at least It is a kind of that 5- hydroxypentanoic acid enzymatics are converted into 1,5- pentanediols with the active polypeptide being selected from the group：Carboxylate reductase activity and Alcohol dehydrogenase activity.Used with the polypeptides in combination with phosphopantetheine transferase reinforcing agent activity and reduced with carboxylic acid The polypeptide of enzymatic activity.Method may further include using it is at least one with the active polypeptide being selected from the group by 1,5- penta 2 Alcohol enzymatic is converted into cadaverine：ω-transaminase activity and alcohol dehydrogenase activity.
In some embodiments, method further includes to use at least one polypeptide with esterase active by glutaric acid Methyl esters enzymatic is converted into glutaric acid.Method may further include using it is at least one with carboxylate reductase activity polypeptide and Glutaric acid enzymatic is converted into 5- aminovaleric acids by least one polypeptide with ω-transaminase activity.In some embodiments, Method further includes to use at least one polypeptide with carboxylate reductase activity and at least one with EC 1.1.1.- Glutaric acid enzymatic is converted into 5- hydroxypentanoic acids by the polypeptide of the dehydrogenase activity of classification.Can with phosphopantetheine The polypeptides in combination of transferase reinforcing agent activity uses the polypeptide with carboxylate reductase activity.
Polypeptide with esterase active can be with SEQ ID NO:The amino acid sequence listed in 16 has at least 70% sequence Row homogeneity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with carboxylate reductase activity can be with SEQ ID NO:The amino acid sequence listed in any one of 2-7 With at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with thioesterase activity can be with SEQ ID NO:The amino acid sequence listed in 17 or 18 has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with phosphopantetheine transferase reinforcing agent activity can be with SEQ ID NO:Arranged in 14 or 15 The amino acid sequence for going out have at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with ω-transaminase activity can be with SEQ ID NO:The amino acid sequence listed in 8-13 has at least 70% sequence identity (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
In another aspect, presents is characterised by the method for generating glutaric acid, and methods described is used including (i) to be had The polypeptide of heptanedioyl-[acp] methyl ester methyl esterase active by glutaryl-[acp] methyl esters enzymatic be converted into glutaryl- [acp] or glutaryl-CoA methyl esters enzymatics are converted into glutaryl-CoA, and (ii) has thioesters using at least one Enzymatic activity, reversible CoA ligase activity, CoA transferase actives, acylated dehydrogenase activity, aldehyde dehydrogenase activity, glutaric acid half Aldehyde dehydrogenase activity, or the polypeptide of succinic semialdehyde dehydrogenase activity turns glutaryl-[acp] or glutaryl-CoA enzymatics Turn to glutaric acid.In some embodiments, using the polypeptide with thioesterase activity by glutaryl-[acp] or glutaryl Base-CoA enzymatics are converted into glutaric acid.In some embodiments, shifted using with reversible CoA ligase activity or CoA Glutaryl-[acp] or glutaryl-CoA enzymatics are converted into glutaric acid by the polypeptide of enzymatic activity.In some embodiments, Using with acylated dehydrogenase activity, aldehyde dehydrogenase activity, glutarate-semialdehyde dehydrogenase activity, or succinic semialdehyde dehydrogenase is lived Glutaryl-[acp] or glutaryl-CoA enzymatics are converted into glutaric acid by the polypeptide of property.
In some embodiments, with heptanedioyl-polypeptide of [acp] methyl ester methyl esterase active can be with SEQ ID NO:The amino acid sequence listed in 1 have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%).
In another aspect, presents is characterised by recombinant host cell.Recombinant host cell is compiled comprising at least one Code has the polypeptide of malonyl-CoA O- methyl transferase activities and the exogenous nucleic acid of the polypeptide with thioesterase activity, institute State host's generation glutaric acid methyl esters.Host can further comprising the allogenic polypeptide with carboxylate reductase activity, the host Further generate glutaric acid semialdehyde methyl esters.Can be with the polypeptides in combination with phosphopantetheine transferase reinforcing agent activity Use the polypeptide with carboxylate reductase activity.
In some embodiments, host further has the active external source being selected from the group many comprising one or more Peptide：Synthase activity, dehydrogenase activity, Dehydratase activity, and reductase activity.
In some embodiments, host further has the active external source being selected from the group many comprising one or more Peptide：Synthase activity, beta-Ketothiolase activity, dehydrogenase activity, hydratase activity, and reductase activity.
Polypeptide with malonyl-CoA O- methyl transferase activities can be with SEQ ID NO:The amino listed in 21 Acid sequence have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
The polypeptide with thioesterase activity can be with SEQ ID NO:The amino acid sequence listed in any one of 17-18 Row have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with reductase activity can be with SEQ ID NO:The amino acid sequence listed in 19 or 20 has at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
In some embodiments, host is further comprising the allogenic polypeptide with esterase active, and the host is further Generation glutaric acid or 5- oxopentanoic acids.Host can also include the allogenic polypeptide with ω-transaminase activity, host's generation 5- aminovaleric acids.
In some embodiments, host is also comprising with the one or more active allogenic polypeptide being selected from the group：ω- Transaminase activity, carboxylate reductase activity and esterase active, the host generate 5- aminovaleric acids.
In some embodiments, host also has the active allogenic polypeptide being selected from the group comprising one or more：Carboxylic Sour reductase activity, N- acetyl transferase activities and deacetylase activity, the host generate cadaverine from 5- aminovaleric acids.
In some embodiments, host also has the active allogenic polypeptide being selected from the group comprising one or more：Carboxylic Sour reductase activity and ω-transaminase activity, the host generate cadaverine from 5- oxopentanoic acids.
In some embodiments, host also has the active allogenic polypeptide being selected from the group comprising one or more：Ester Enzymatic activity, 6 hydroxycaproic acid dehydrogenase activity, 4 hydroxybutyric acid dehydrogenase activity, 5- hydroxypentanoic acid dehydrogenase activities, and alcohol take off Hydrogenase activity, the host generates 5- hydroxypentanoic acids.Comprising one or more with the active allogenic polypeptide being selected from the group Host can generate 1,5- pentanediols from 5- hydroxypentanoic acids：Carboxylate reductase activity and alcohol dehydrogenase activity.Comprising a kind of or many The host that planting has the active allogenic polypeptide being selected from the group can generate cadaverine from 1,5- pentanediols：Alcohol dehydrogenase activity and ω-transaminase activity.The host comprising one or more with the active allogenic polypeptide being selected from the group can be from 5- hydroxyls penta Acid generation cadaverine：Carboxylate reductase activity, alcohol dehydrogenase activity and ω-transaminase activity.
In some embodiments, host comprising with carboxylate reductase activity allogenic polypeptide when, it with have phosphorus The allogenic polypeptide of sour pantetheine transferase reinforcing agent activity is applied in combination.
Polypeptide with carboxylate reductase can be with SEQ ID NO:The amino acid sequence that any one of 2-7 is listed has extremely Few 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with ω-transaminase activity can be with SEQ ID NO:The amino acid that any one of 8-13 is listed has extremely Few 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
Polypeptide with phosphopantetheine transferase reinforcing agent activity can be with SEQ ID NO:Arranged in 14 or 15 The amino acid sequence for going out have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
In another aspect, presents is characterised by recombinant host, and it includes at least one exogenous nucleic acid, the external source Nucleic acid coding has the polypeptide of heptanedioyl-[acp] methyl ester methyl esterase active, and at least one active with what is be selected from the group Polypeptide：Thioesterase activity, reversible CoA ligase activity, CoA transferase actives, acylated dehydrogenase activity, aldehyde dehydrogenase is lived Property, glutarate-semialdehyde dehydrogenase activity, and succinic semialdehyde dehydrogenase activity.In some embodiments, with heptanedioyl- The polypeptide of [acp] methyl ester methyl esterase active and SEQ ID NO:The amino acid sequence listed in 1 has at least 70% sequence same One property (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)
In another aspect, presents is characterised by the method for generating 5 biologically-derived carbon compounds.For giving birth to Method into 5 biologically-derived carbon compounds can include under certain conditions cultivating or raw host as described in this article Enough time periods are grown to generate the 5 biologically-derived carbon compounds, wherein optionally, the 5 biologically-derived carbon compounds Thing is selected from the group：5- oxopentanoic acids, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine, 1,5-PD, and combinations thereof.
In an aspect, presents is characterised by composition, and it includes 5 biologically-derived as described in this article carbonizations Compound and the compound different from the 5 biologically-derived carbon compounds, wherein the 5 biologically-derived carbon compounds are selected from down Group：5- oxopentanoic acids, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine, 1,5-PD, and combinations thereof.For example, biologically-derived 5 Carbon compound is the cellular portions of host cell or organism.
Presents is further characterized in that the polymer based on biological (biobased polymer), and it includes biologically-derived 5- Oxopentanoic acid, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine, 1,5-PD, and combinations thereof.
Presents is further characterized in that comprising biologically-derived 5- oxopentanoic acids, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine, 1,5-PD, and combinations thereof the molded product obtained based on biological resin based on biological resin and by molding.
In another aspect, presents is characterised by that it is included in poly- for generating the method based on biological resin Make the biologically-derived 5- oxopentanoic acids, 5- hydroxypentanoic acids, 5- aminovaleric acids, cadaverine, 1,5- penta 2 in compound reaction of formation Alcohol and itself or another compound chemically reactive.
In another aspect, presents is characterised by biologically-derived product (bio-derived product), is based on The biological derivative product (fermentation-derived product) of product (bio-based product) or fermentation, Wherein described product is included：(i.) composition, the composition includes at least one biologically-derived, base as described in this article In the biological or derivative compound of fermentation, or its any combinations；(ii.) biologically-derived, based on biology or fermentation derives Polymer, its include (i.) it is biologically-derived, based on biological or the derivative composition of fermentation or compound, or its is any Combination；(iii.) it is biologically-derived, based on the biological or derivative resin of fermentation, its include (i.) it is biologically-derived, be based on The derivative compound of biological or fermentation or biologically-derived, based on biology or the derivative composition of fermentation or its any combinations Or biologically-derived, based on biology or the derivative polymer of fermentation or its any combinations of ii.；(iv.) molding substance, it leads to Cross make (ii.) it is biologically-derived, based on the biological or derivative polymer of fermentation or (iii.) it is biologically-derived, based on life Thing or the derivative resin of fermentation or its any combinations molding obtain；(v.) biologically-derived, based on biology or fermentation derives Preparaton, its include (i.) it is biologically-derived, based on the biological or derivative composition of fermentation, (i.) it is biologically-derived , based on the biological or derivative compound of fermentation, (ii.) it is biologically-derived, based on the biological or derivative polymerization of fermentation Thing, (iii.) be biologically-derived, based on the biological or derivative resin of fermentation, or (iv.) it is biologically-derived, based on biological Or the derivative molding substance of fermentation, or its any combinations；Or (vi.) is biologically-derived, based on biology or fermentation is derivative partly Solid or non-semisolid flow (stream), its include (i.) it is biologically-derived, based on the biological or derivative composition of fermentation, (i.) it is biologically-derived, based on the biological or derivative compound of fermentation, (ii.) it is biologically-derived, based on biological or The derivative polymer of fermentation, (iii.) be biologically-derived, based on the biological or derivative resin of fermentation, (v.) biologically-derived, Based on the biological or derivative preparaton of fermentation, or iv it is biologically-derived, based on the biological or derivative molding substance of fermentation, Or its any combinations.
Presents is further characterized in that and improves the polypeptide with carboxylate reductase activity to substituted or unsubstituted C4-C8Two The active method of carboxylic acid such as glutaric acid or adipic acid.Method is included using with malonyl-CoA methyl transferase activities Polypeptide by the C4-C8Dicarboxylic acids enzymatic is converted into HOC (=O) (C2-C6Alkyl (alkyl))-C (=O) OCH3Ester, makes afterwards With the polypeptide with carboxylate reductase activity by HOC (=O) (C2-C6Alkyl)-C (=O) OCH3Esterase promotees to be converted into HC (=O) (C2-C6Alkyl)-C (=O) OCH3.Method further can include using the polypeptide with dehydrogenase activity by the HC (=O) (C2-C6Alkyl)-C (=O) OCH3It is converted into HOCH2(C2-C6 alkyl)-C (=O) OCH3.In some embodiments, method is entered One step includes using the polypeptide with esterase active by HOCH2(C2-C6Alkyl)-C (=O) OCH3Enzymatic is converted into HOCH2(C2- C6Alkyl)-C (=O) OH.
Biochemistry network is described herein, its include malonyl-CoA O- transmethylases and malonyl- At least one in [acp] and malonyl-CoA, wherein malonyl-CoA O- transmethylases are by malonyl-[acp] Glutaric acid methyl esters is converted into at least one enzymatic in malonyl-CoA.Biochemistry network is further reduced comprising carboxylic acid Glutaric acid methyl esters enzymatic is converted into glutaric acid semialdehyde methyl esters by enzyme, wherein carboxylate reductase.
In some embodiments, biochemistry network comprising malonyl-CoA O- transmethylases and malonyl- Malonyl-[acp] enzymatic is converted into malonyl-[acp] first by [acp], wherein malonyl-CoA O- transmethylases Ester.Biochemistry network can further include at least one enzyme being selected from the group：Synthase, dehydrogenase, dehydratase, and reduction Enzyme, wherein synthase, dehydrogenase, dehydratase, or reductase by malonyl-[acp] methyl esters enzymatic be converted into glutaryl- [acp] methyl esters.Biochemistry network further includes thioesterase, and wherein thioesterase converts glutaryl-[acp] methyl esters enzymatic It is glutaric acid methyl esters.
In some embodiments, biochemistry network comprising malonyl-CoA O- transmethylases and malonyl- Malonyl-CoA enzymatics are converted into malonyl-CoA methyl esters by CoA, wherein malonyl-CoA O- transmethylases.It is raw Thing chemical network can further include at least one enzyme being selected from the group：Synthase, beta-Ketothiolase, dehydrogenase, hydrase, and Be converted into for malonyl-CoA methyl esters enzymatics by reductase, wherein synthase, beta-Ketothiolase, dehydrogenase, hydrase, and reductase Glutaryl-CoA methyl esters.Biochemistry network can further include thioesterase, and wherein thioesterase is by glutaryl-CoA first Esterase promotees to be converted into glutaric acid methyl esters.
In some embodiments, biochemistry network further includes esterase and glutaric acid semialdehyde methyl esters, wherein esterase Glutaric acid semialdehyde methyl esters enzymatic is converted into 5- oxopentanoic acids.Biochemistry network can further include ω-transaminase, wherein 5- oxopentanoic acid enzymatics are converted into 5- aminovaleric acids by ω-transaminase.
In some embodiments, biochemistry network further includes ω-transaminase and glutaric acid semialdehyde methyl esters, wherein Glutaric acid semialdehyde methyl esters enzymatic is converted into 5- aminopentanoic acid methyl esters by ω-transaminase.Biochemistry network can be included further 5- aminopentanoic acid methyl ester enzymatics are converted into 5- aminovaleric acids by esterase, wherein esterase.
In some embodiments, biochemistry network can further include at least one enzyme being selected from the group：Carboxylic acid Be converted into for 5- aminovaleric acid enzymatics by reductase and ω-transaminase and 5- aminovaleric acids, wherein carboxylate reductase and ω-transaminase Cadaverine.
In some embodiments, biochemistry network can further include at least one enzyme being selected from the group and 5- ammonia Base valeric acid：N- acetyltransferases, carboxylate reductase, ω-transaminase, and deacetylase, wherein N- acetyltransferases, carboxylic 5- aminovaleric acid enzymatics are converted into cadaverine by sour reductase, ω-transaminase, and deacetylase.
In some embodiments, biochemistry network can further include at least one enzyme being selected from the group and 5- oxygen For valeric acid：Be converted into for 5- oxopentanoic acid enzymatics by carboxylate reductase and ω-transaminase, wherein carboxylate reductase and ω-transaminase Cadaverine.
In some embodiments, biochemistry network can further include at least one enzyme being selected from the group and penta 2 Sour semialdehyde methyl esters：Esterase and 6 hydroxycaproic acid dehydrogenase, wherein esterase and 6 hydroxycaproic acid dehydrogenase are by glutaric acid semialdehyde methyl esters Enzymatic is converted into 5- hydroxypentanoic acids.
In some embodiments, biochemistry network can further include at least one enzyme being selected from the group and penta 2 Sour semialdehyde methyl esters：Glutaric acid semialdehyde methyl esters enzymatic is converted into 5- hydroxyls by esterase and alcohol dehydrogenase, wherein esterase and alcohol dehydrogenase Valeric acid.
In some embodiments, biochemistry network can further include alcohol dehydrogenase, and 5- hydroxypentanoic acids, wherein 5- hydroxypentanoic acid enzymatics are converted into 1,5- pentanediols by carboxylate reductase and alcohol dehydrogenase.Biochemistry network can be wrapped further Containing at least one enzyme being selected from the group：ω-transaminase and alcohol dehydrogenase, wherein ω-transaminase and alcohol dehydrogenase are by 1,5- penta 2 Alcohol enzymatic is converted into cadaverine.
In some embodiments, biochemistry network can further include at least one enzyme being selected from the group and 5- hydroxyls Base valeric acid：Carboxylate reductase, ω-transaminase and alcohol dehydrogenase, wherein carboxylate reductase, ω-transaminase and alcohol dehydrogenase are by 5- Hydroxypentanoic acid enzymatic is converted into cadaverine.
Presents is further characterized in that recombinant host, and it includes at least one exogenous nucleic acid, and the exogenous nucleic acid encodes (i) third Diacyl-[acp] O- transmethylases, (ii) heptanedioyl-[acp] methyl ester methyl esterase and (iii) thioesterase, and generate penta Carbomethoxyphenyl, glutaryl-[acp] or glutaryl-CoA.
The such recombinant host for generating glutaric acid methyl esters can further include esterase, and further generate glutaric acid.
Generating such recombinant host of glutaryl-[acp] can further include thioesterase and generate glutaric acid.
Such recombinant host of generation glutaryl-CoA can further be included following one or more：(i) thioesterase, (ii) reversible CoA ligase, (iii) CoA- transferases, or (iv) is acylated dehydrogenase, and (v) aldehyde dehydrogenase, such as 7- oxos Enanthic acid dehydrogenase, 6- oxo caproic acid dehydrogenases or 5- oxopentanoic acids dehydrogenase and further generation glutaric acid or 5- oxos penta Acid.
The recombinant host of generation 5- oxopentanoic acids or glutaric acid can further include one or more of：(i) ω-turn Ammonia enzyme or (ii) carboxylate reductase, and further generate 5- aminovaleric acids.
The recombinant host for generating glutaric acid methyl esters can further be included following one or more：(i) ω-transaminase or (ii) carboxylate reductase and (iii) esterase and 5- aminovaleric acids are further generated.
The recombinant host of generation 5- oxopentanoic acids or glutaric acid can further include one or more of：I () alcohol takes off Hydrogen enzyme or (ii) carboxylate reductase, and further generate 5- hydroxypentanoic acids.
The recombinant host for generating glutaric acid methyl esters can further be included following one or more：(i) alcohol dehydrogenase, (ii) Esterase or (iii) carboxylate reductase and further generate 5- hydroxypentanoic acids.
The recombinant host for generating 5- hydroxypentanoic acids can further include one or more of：(i) carboxylase reductase (ii) alcohol dehydrogenase, the host further generates 1,5-PD.
The recombinant host for generating 5- hydroxypentanoic acids can further include one or more of：(i) carboxylase reductase, (ii) one or more ω-transaminase and (iii) alcohol dehydrogenase, the host further generate cadaverine.
The recombinant host for generating 5- aminovaleric acids can further include one or more of：(i) carboxylase reductase (ii) ω-transaminase, the host further generates cadaverine.
The recombinant host for generating 5- oxopentanoic acids can further include one or more of：(i) carboxylase reductase (ii) one or more ω-transaminase, the host further generates cadaverine.
The recombinant host for generating 1,5- pentanediols can be further a kind of comprising (i) one or more alcohol dehydrogenase and (ii) Or various ω-transaminases, the host further generates cadaverine.
The recombinant host for generating 5- aminovaleric acids can further include one or more of：(i) N- acetyl grouptransfers Enzyme, (ii) carboxylate reductase, (iii) ω-transaminase and (iv) acetyltransferase, the host further generate cadaverine.
In any embodiment being described herein, recombinant host can be prokaryotes, such as from Escherichia Category (Escherichia) such as Escherichia coli (Escherichia coli)；From fusobacterium (Clostridia) such as Young Clostridium (Clostridium ljungdahlii), from producing and ethanol clostridium (Clostridium autoethanogenum) or Crewe Buddhist clostridium (Clostridium kluyveri)；From corynebacterium (Corynebacteria) such as corynebacterium glutamicum (Corynebacterium glutamicum)；From greedy copper Pseudomonas (Cupriavidus) such as hookworm corrupt bacteria (Cupriavidus necator) or resistance to metal covet copper bacterium (Cupriavidus metallidurans)；From pseudomonas (Pseudomonas) such as Pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida) or Pseudomonas oleovorans (Pseudomonas oleavorans)；From acidophilic bacteria (Delftia acidovorans), from bacillus (Bacilluss) such as Bacillus subtillis (Bacillus subtillis)；From lactobacillus (Lactobacillus) such as Lactobacillus delbrueckii (Lactobacillus delbrueckii)；From lactococcus (Lactococcus) such as Lactococcus lactis (Lactococcus lactis) or next From Rhod (Rhodococcus) such as Rhodococcus equi (Rhodococcus equi).
In any embodiment being described herein, recombinant host can be eucaryote, such as from aspergillus (Aspergillus) such as aspergillus niger (Aspergillus niger)；Such as made wine from saccharomyces (Saccharomyces) Yeast (Saccharomyces cerevisiae)；Belong to (Pichia) such as pichia pastoris phaff from complete Chi Shi ferment (Pichia pastoris)；From Ye Luoweiya saccharomyces (Yarrowia) such as Yarrowialipolytica (Yarrowia Lipolytica), from Issatchenkia (Issatchenkia) such as Issatchenkia orientalis (Issathenkia Orientalis), from the inferior Dbaly yeast (Debaryomyces of Debaryomyces (Debaryomyces) such as Chinese Hansenii), such as Arxula adenoinivorans are belonged to from Arxula, or from Kluyveromyces (Kluyveromyces) the such as eucaryote of lactic acid yeast kluyveromyces (Kluyveromyces lactis).
In some embodiments, weaken or the endogenous biological chemical network of lifting host ensures acetyl-CoA with (1) Intracellular availability, (2) create that co-factor (i.e. NADH or NADPH) is uneven, and it can be balanced via the formation of C5 building blocks, (3) prevent to cause and the central metabolites thing comprising C5 building blocks, the degraded of center precursor and (4) are ensured from the effective of cell Outflow.
Any method can be carried out in recombinant host by fermenting.In some embodiments, host undergo it is aerobic or Training strategy under micro- aerobic condition of culture.Host can undergo aerobic, the training strategy under anaerobism or micro- aerobic condition of culture. Can be limited in nutrition, host is cultivated under conditions of such as phosphate, oxygen or nitrogen limitation.The place can be retained using ceramic membrane Lead to maintain high-cell density during fermentation.
In some embodiments, biological raw material can be used as the primary carbon source of fermentation.For example, biological raw material can be Or monose, disaccharides, lignocellulosic, hemicellulose, cellulose, lignin, levulic acid and formic acid, glycerine three can be derived from Ester, glycerine, aliphatic acid, agricultural wastes, concentration vinasse (condensed distillers'solubles) or municipal waste.
In some embodiments, abiotic raw material can serve as the primary carbon source of fermentation.Abiotic raw material can be or Natural gas, synthesis gas, CO can be derived from2/H2, methyl alcohol, ethanol, benzoate (ester), non-volatile residue (NVR) or come from Alkali wash water (caustic wash) waste stream of cyclohexane oxidation process, terephthalic acid (TPA)/isophathalic acid mixture waste stream.
In some embodiments, host shows the tolerance to the C5 building blocks of high concentration.In some embodiments In, improve the tolerance to the C5 building blocks of high concentration via the continuous culture in selective environment.
In some embodiments, host includes the decrease with the one or more active polypeptide being selected from the group：It is poly- Hydroxy alkanoic acid ester synthase, acetyl-CoA thioesterase, acetyl-CoA specificity beta-Ketothiolase, acetoacetyl-CoA reduction Enzyme, the phosphoric acid acetic acid transferase, acetokinase, lactic dehydrogenase, the menaquinol- fumaric acid redox that form acetic acid Enzyme, 2- ketone acids (oxoacid) decarboxylase for producing isobutanol, alcohol dehydrogenase, triose-phosphate isomerase, the pyruvic acid for forming ethanol It is decarboxylase, GPI, the unbalanced transhydrogenase of dissipation co-factor, special to creating unbalanced co-factor Glutamte dehydrogenase, the glutamte dehydrogenase using NADH/NADPH, the heptanedioyl-CoA dehydrogenases of property；Receive C5 building blocks and Center precursor as substrate acyl-CoA dehydrogenase；Glutaryl-CoA dehydrogenases；With heptanedioyl-CoA synzyme.
Any recombinant host described herein further can be described with the gene of one or more coded polypeptide of overexpression Polypeptide has：Acetyl-CoA synzyme.6-phosphogluconate dehydrogenase；Transketolase；Feed back resistance threonine deaminase； Pyridine nucleotide transhydrogenase；Hydrogenlyase；Glyceraldehyde -3P- dehydrogenases；Malate dehydrogenase；Glucose-6-phosphate dehydrogenase (G6PD)；Really Sugared 1,6 diphosphatases；Propiono-CoA synzyme；L-alanine dehydrogenase；Pidolidone dehydrogenase；Glu synthesizes Enzyme；Lysine transport protein；Dicarboxylic acids transport protein；And/or multidrug transporter activity.
The reaction of approach specifically described herein can be carried out in one or more cell (such as host cell) bacterial strain, described Bacterial strain (a) naturally expresses one or more relevant enzyme, and (b) transforms to express one or more relevant enzyme through genetically engineered, or C () naturally expresses one or more relevant enzyme and genetically engineered transformation is to express one or more relevant enzyme.Or, can be with Relevant enzyme is extracted from the host cell of the above-mentioned type, and is used with purifying or half purified form.The enzyme of extraction can be optionally It is fixed on the bottom of suitable reaction vessel and/or wall.Additionally, these extracts include can be used as the cracking of related enzyme source Thing (such as cell lysate).In the method that document is provided, all steps can be carried out in cell (such as host cell), All steps can be carried out using the enzyme for extracting, or some steps can be carried out in cell, and other steps can be used The enzyme of extraction is carried out.
It will be appreciated by those skilled in the art that when the acid proton being present in parent compound is by metal ion, such as alkali gold Category ion, alkaline-earth metal ions or aluminium ion are substituted；Or when being coordinated with organic base, compound containing hydroxy-acid group (including but It is not limited to organic acid, carboxylic acid, amino acid and dicarboxylic acids) formed or change into its ion salt form.It is acceptable organic Alkali includes but is not limited to monoethanolamine, diethanol amine, triethanolamine, tromethamine, N-METHYL-ALPHA-L-GLUCOSAMINE etc..Acceptable inorganic base Including but not limited to aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, NaOH etc..Salt of the invention is separated into salt Or change into free acid by via adding acid or pH being down into below pKa with acid-exchange resin treatment.
It will be appreciated by those skilled in the art that compound (including but not limited to organic amine, amino acid and two containing amine groups Amine) formed or be converted into their ion salt form, ammonium salt for example is formed by addition acid proton in amine, with inorganic acid such as Hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc. are formed；Or formed with organic acid, including but not limited to acetic acid, propionic acid, caproic acid, ring Pentane propionic acid, glycolic, pyruvic acid, lactic acid, malonic acid, butanedioic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, Benzoic acid, 3- (4- hydroxy benzoyls) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, 1,2- ethionic acid, 2- hydroxyls Base ethyl sulfonic acid, benzene sulfonic acid, 2- naphthalene sulfonic acids, 4- methyl bicycles-[2.2.2] oct-2-ene -1- carboxylic acids, glucoheptonic acid, 4,4'- methylenes Base pair-(3- hydroxyl -2- alkene -1- carboxylic acids), 3- phenylpropionic acids, trimethylace tonitric, butylacetic acid, lauryl sulfate, gluconic acid, Glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid etc..Acceptable inorganic base includes but is not limited to aluminium hydroxide, Calcium hydroxide, potassium hydroxide, sodium carbonate, NaOH etc..Salt of the invention is separated or by via addition alkali in a salt form Or pH is extremely changed into unhindered amina higher than pKb with deacidite treatment.
It will be appreciated by those skilled in the art that compound (the including but not limited to amino containing both amine groups and hydroxy-acid group Acid) pass through formed below or change into their ion salt form：It is described inorganic by the acid-addition salts for 1) being formed with inorganic acid Acid includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.；Or the acid-addition salts formed with organic acid, it is described organic Acid includes but is not limited to acetic acid, propionic acid, caproic acid, pentamethylene propionic acid, glycolic, pyruvic acid, lactic acid, malonic acid, butanedioic acid, apple Acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4- hydroxy benzoyls) benzoic acid, cinnamic acid, mandelic acid, Methanesulfonic acid, ethyl sulfonic acid, 1,2- ethionic acid, 2- ethylenehydrinsulfonic acids, benzene sulfonic acid, 2- naphthalene sulfonic acids, 4- methyl bicycles-[2.2.2] octyl- 2- alkene -1- carboxylic acids, glucoheptonic acid, 4,4'- di-2-ethylhexylphosphine oxides-(3- hydroxyl -2- alkene -1- carboxylic acids), 3- phenylpropionic acids, trimethylace tonitric Acid, butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid etc..Can connect The inorganic base received including but not limited to aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, NaOH etc., or 2) work as presence When acid proton in parent compound is replaced by metal ion, such as alkali metal ion, alkaline-earth metal ions or aluminium ion； Or be coordinated with organic base.Acceptable organic base includes but is not limited to monoethanolamine, diethanol amine, triethanolamine, tromethamine, N- Methylglucosamine etc..Acceptable inorganic base includes but is not limited to aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, hydrogen-oxygen Change sodium etc..Salt of the invention can as salinity from or by via addition acid or with acid-exchange resin process pH is dropped Free acid is converted into below pKa.
Unless otherwise defined, all technologies used herein and scientific terminology and ordinary skill of the art Personnel's is generally understood that with identical implication.Although the method similar or equivalent with method and material specifically described herein and material Material can be used to implement the present invention, but described below is suitable method and material.All publications mentioned in this article, patent Shen Please, patent and other bibliography, including GenBank the and NCBI submission with accession number are integrally incorporated by reference. In the case of conflict, it is defined by this specification (including definition).Additionally, material, the method and embodiment are merely illustrative, Rather than restricted.
The details of one or more embodiments of the invention is elaborated in following accompanying drawing and description.It is of the invention other Feature, objects and advantages will be apparent from specification, drawings and the claims book.Standard reality in Patent Law Trample, word in claims " including " can be substituted by " substantially by ... constitute " or by " consist of ".
Brief description
Fig. 1 is to cause the exemplary bio chemistry way of glutaric acid methyl esters or glutaryl-[acp] from malonyl-[acp] The schematic diagram in footpath.
Fig. 2 is to cause glutaric acid methyl esters or glutaryl-CoA from malonyl-CoA using NADPH as reducing equivalent Exemplary bio chemistry route schematic diagram.
Fig. 3 is to cause glutaric acid methyl esters or glutaryl-CoA from malonyl-CoA using NADH as reducing equivalent The schematic diagram of exemplary bio chemistry route.
Fig. 4 is to use glutaric acid methyl esters, and glutaryl-[acp] or glutaryl-CoA cause penta 2 as center precursor The schematic diagram of the exemplary bio chemistry route of acid.
Fig. 5 is to cause the exemplary bio chemistry of 5- aminovaleric acids as center precursor using glutaric acid methyl esters or glutaric acid The schematic diagram of approach.
Fig. 6 is that, using 5- aminovaleric acids, 5- hydroxypentanoic acids, or 1,5-PD causes the example of cadaverine as center precursor The schematic diagram of property bio-chemical pathway.
Fig. 7 is to cause the exemplary bio chemistry way of cadaverine as center precursor using glutaric acid semialdehyde or 5- aminovaleric acids The schematic diagram in footpath.
Fig. 8 is to cause the exemplary bio chemistry of 5- hydroxypentanoic acids as center precursor using glutaric acid methyl esters or glutaric acid The schematic diagram of approach.
Fig. 9 is to cause showing for the exemplary bio chemistry route of 1,5 pentanediols as center precursor using 5- hydroxypentanoic acids It is intended to.
Figure 10 contain Escherichia coli heptanedioyl-[acp] methyl ester methyl esterase (referring to Genbank accession number AAC76437.1, SEQ ID NO:1), Mycobacterium marinum (Mycobacterium marinum) carboxylate reductase is (referring to Genbank accession number ACC40567.1, SEQ ID NO:2), mycobacterium smegmatis (Mycobacterium smegmatis) carboxylate reductase (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Segniliparus rugosus carboxylate reductases (referring to Genbank accession number EFV11917.1, SEQ ID NO:4), mycobacterium smegmatis carboxylate reductase is (referring to Genbank accession number ABK75684.1, SEQ ID NO:5), Marseille mycobacteria (Mycobacterium massiliense) carboxylate reductase (ginseng See Genbank accession number EIV11143.1, SEQ ID NO:6), Segniliparus rotundus carboxylate reductases (referring to Genbank accession number ADG98140.1, SEQ ID NO:7), blue or green chromabacterium biolaceum (Chromobacterium violaceum) ω- Transaminase is (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa (Pseudomonas Aeruginosa) ω-transaminase is (referring to Genbank accession number AAG08191.1, SEQ ID NO:9), pseudomonas syringae (Pseudomonas syringae) ω-transaminase is (referring to Genbank accession number AAY39893.1, SEQ ID NO:10), ball The red bacillus of shape (Rhodobacter sphaeroides) ω-transaminase is (referring to Genbank accession number ABA81135.1, SEQ ID NO:11), Escherichia coli ω-transaminase is (referring to Genbank accession number AAA57874.1, SEQ ID NO:12), vibrio fluvialis (Vibrio fluvialis) ω-transaminase is (referring to Genbank accession number AEA39183.1, SEQ ID NO:13), withered grass bud Spore bacillus Phosphopantetheinyl transferase is (referring to Genbank accession number CAA44858.1, SEQ ID NO:14), promise card Salmonella species (Nocardia sp.) Phosphopantetheinyl transferases of NRRL 5646 are (referring to Genbank accession number ABI83656.1, SEQ ID NO:15), Pseudomonas fluorescens esterase is (referring to Genbank accession number AAC60471.2, SEQ ID NO:16), Lactobacillus brevis acyl group-[acp] thioesterase is (referring to Genbank accession number ABJ63754.1, SEQ ID NO:17), plant Thing lactobacillus acyl group-[acp] thioesterase is (referring to Genbank accession number ABJ63754.1, SEQ ID NO:18), the close spiral of tooth dirt Body (Treponema denticola) enoyl-CoA reductase (see, for example, Genbank accession number AAS11092.1, SEQ ID Nos:19), Euglena gracilis (Euglena gracilis) enoyl-CoA reductase (see, for example, Genbank accession number AAW66853.1,SEQ ID No:20), Bacillus cercus (Bacillus cereus) malonyl-[acp] O- methyl turns Move enzyme and (see, for example, Genbank accession number AAP11034.1, SEQ ID Nos:21), Escherichia coli thioesterase (see e.g., Genbank accession number AAB59067.1SEQ ID NO:, and Escherichia coli thioesterase (Genbank accession number 22) AAA24665.1,SEQ ID NO:23) amino acid sequence.
Figure 11 is the block diagram of percentage conversion (mol/mol) behind pyruvic acid to 4 hours of ALANINE, used as relative In empty vector control, the ω-transaminase activity of the 4 kinds of ω-transaminase prepared product for cadaverine to be converted into 5- amino valerals is surveyed Amount.
Figure 12 is block diagram, its summarize 20 minutes after absorbance change at 340nm, its be only enzyme control it is (bottomless Thing) in NADPH consumption and the active measurements of 5 kinds of carboxylate reductase prepared products.
Figure 13 is the block diagram of percentage conversion (mol/mol) behind pyruvic acid to 4 hours of ALANINE, used as relative In empty vector control, the ω-transaminase of the 6 kinds of ω-transaminase prepared product for 5- aminopentanols to be converted into 5- oxo amylalcohols Activity measurement.
Figure 14 is the block diagram of the absorbance change at 340nm after 20 minutes, and it is relative to empty vector control, NADPH Consumption and the active measurement for 5- hydroxypentanoic acids to be converted into 5 kinds of carboxylate reductase prepared products of 5- hydrogenation of hydroxypentylaldehyd.
Figure 15 is the block diagram of percentage conversion (mol/mol) behind pyruvic acid to 4 hours of ALANINE, used as relative In empty vector control, for by N5- acetyl group -1,5- 1,5-DAPs be converted into 5 kinds of ω of N5- acetyl group -5- amino valerals - The ω of transaminase prepared product-transaminase activity measurement.
Figure 16 is the block diagram of the change of the absorbance at the 340nm after 20 minutes, its be relative to empty vector control, The consumption of NADPH and the active measurement for glutaric acid semialdehyde to be converted into the carboxylate reductase prepared product of glutaraldehyde.
Figure 17 is block diagram, the third of its measurement for summarizing the ω-transaminase activity as the control (without substrate) of only enzyme Ketone acid to the percentage of ALANINE converts (mol/mol).
Figure 18 is the block diagram of percentage conversion (mol/mol) behind pyruvic acid to 4 hours of ALANINE, used as relative In empty vector control, the ω-transaminase of a kind of ω-transaminase prepared product for 5- aminovaleric acids to be converted into glutaric acid semialdehyde The measurement of activity.
Figure 19 is the block diagram of percentage conversion (mol/mol) behind ALANINE to 4 hours of pyruvic acid, used as relative In empty vector control, the ω-transaminase of a kind of ω-transaminase prepared product for glutaric acid semialdehyde to be converted into 5- aminovaleric acids The measurement of activity.
Figure 20 is the block diagram of the change of the absorbance at the 340nm after 20 minutes, its be relative to empty vector control, The consumption of NADPH and the active survey for glutaric acid methyl esters to be converted into the carboxylate reductase prepared product of glutaric acid semialdehyde methyl esters Amount.
Figure 21 is the block diagram of percentage conversion (mol/mol) behind ALANINE to 4 hours of pyruvic acid, used as relative In empty vector control, the ω-transaminase activity of a kind of ω-transaminase prepared product for 1- aminopentanes to be converted into valeral Measurement.
Figure 22 is the block diagram of percentage conversion (mol/mol) behind ALANINE to 4 hours of pyruvic acid, used as relative In empty vector control, the ω-transaminase activity of a kind of ω-transaminase prepared product for 1- aminoheptanes to be converted into enanthaldehyde Measurement.
Figure 23 be by heptanedioyl-[acp] methyl ester methyl esterase by glutaryl-CoA methyl esters be converted into glutaryl- Table behind 1 hour of CoA.
Detailed description of the invention
Presents provides enzyme, non-native pathway, training strategy, raw material, host microorganism and the biochemistry to host The decrease of network, it produces 5 carbon backbones from center metabolin, and such as glutaryl-CoA or 5- oxopentanoic acids (are also called penta 2 Sour semialdehyde), wherein one or more functional end-groups can be formed, cause glutaric acid, 5- aminovaleric acids, cadaverine (to be also called 1,5 Pentanediamine), 5- hydroxypentanoic acids, or one or more of 1,5-PD (hereinafter " C5 building blocks ") of synthesis.Can give birth to Into glutaric acid semialdehyde (being also called 5- oxopentanoic acids) as the intermediate of other products.As used herein, term is " before center Body " is used to mean to cause any metabolin in any metabolic pathway shown herein of C5 building blocks synthesis.Term " center Metabolin " is used to mean to generate to support the metabolin of growth in all microorganisms.
Host microorganism described herein can include endogenous pathway, and the endogenous pathway can be by operation so that can To generate one or more C5 building block.In endogenous pathway, host microorganism naturally expresses the institute of the reaction in catalytic route There is enzyme.Host microorganism containing engineering approach does not express all enzymes of the reaction in catalytic route naturally, but has passed through Cross engineered so that all enzymes in expression approach in host.
Nucleic acid (or protein) and host use as referred to herein, and term " external source " refers to unlike it quilt in nature It was found that the nucleic acid of (and can not be obtained from specific cell type) or being compiled by the nucleic acid in being equally present in specific cell type The protein of code.In this way, non-naturally occurring nucleic acid is once considered as the external source for host in host.It is important that Notice that non-naturally occurring nucleic acid can contain the nucleic acid subsequence or fragment of the nucleotide sequence found in nature, as long as the core Acid is not present in nature as overall.For example, the nucleic acid molecules that expression vector is contained within genomic dna sequence are non-naturals The nucleic acid of presence, so once it for host cell is external source to import in host, because the nucleic acid molecules are used as entirety (genomic DNA plus vector DNA) is not present in nature.In this way, any carrier being not present in as entirety in nature, The plasmid or virus (such as retrovirus, adenovirus or herpesviral) of autonomous replication are considered as non-naturally occurring nucleic acid.By This genomic DNA fragment and cDNA produced by PCR or limitation inscribe nucleic acid ferment treatment of drawing a conclusion is also considered as non-day The nucleic acid for so existing, because they exist as the separate molecule for being not found in nature.It also follows that any with not See any nucleic acid that the arrangement in nature contains promoter sequence and polypeptid coding sequence (such as cDNA or genomic DNA) It is also non-naturally occurring nucleic acid.Naturally occurring nucleic acid can be the external source for specific host microorganism.For example, from The complete chromosome of the cell separation of yeast x Yi Dan will the chromosome to import in the cell of yeast y for yeast y cells be outer Source nucleic acid.
Comparatively, nucleic acid (such as gene) (or protein) and host use as referred to herein, term is " endogenous " refer in being present in specific host really just as it is found in nature (and can be obtained from specific host) Nucleic acid (or protein).Additionally, the cell of " endogenous expression " nucleic acid (or protein) is found phase just as it in nature The nucleic acid (or protein) is expressed like that with certain types of host.Additionally, " endogenous generation " nucleic acid, protein or other The host of compound generates the nucleic acid, albumen just as its identical certain types of host when being found in nature Matter or compound.
In some embodiments, the compound for being generated according to host and by host, can express with third in host The polypeptide of diacyl-[acp] O- methyl transferase activities.In some embodiments, the change for being generated according to host and by host Compound, can in host express with malonyl-the polypeptide of [acp] O- methyl transferase activities and with carboxylate reductase The polypeptide of activity.In some embodiments, the compound for being generated according to host and by host, can in host express following One or more polypeptide, including with malonyl-polypeptide of [acp] O- methyl transferase activities, with heptanedioyl-[acp] The polypeptide of methyl ester methyl esterase active, the polypeptide with esterase active, the polypeptide with reversible CoA ligase activity has The polypeptide of CoA- transferase actives, the polypeptide with 4 hydroxybutyric acid dehydrogenase activity, with 5- hydroxypentanoic acid dehydrogenase activities Polypeptide, the polypeptide with 6 hydroxycaproic acid dehydrogenase activity, the polypeptide with alcohol dehydrogenase activity is de- with 5- oxopentanoic acids The polypeptide of hydrogenase activity, the polypeptide with 6- oxo caproic acid dehydrogenase activities, 7- oxo-heptanoic acid dehydrogenase activities, with aldehyde dehydrogenation The polypeptide of enzymatic activity, the polypeptide with ω-transaminase activity, and/or the polypeptide with carboxylate reductase activity.In expression carboxylic acid In the recombinant host of reductase, it is also possible to express Phosphopantetheinyl transferase, because it strengthens carboxylate reductase Activity.
For example, presents is characterised by recombinant host, it includes at least one exogenous nucleic acid, the exogenous nucleic acid coding (i) malonyl-[acp] O- transmethylases, (ii) heptanedioyl-[acp] methyl ester methyl esterase and (iii) thioesterase, and Generation glutaric acid methyl esters, glutaryl-[acp] or glutaryl-CoA.
The such recombinant host for generating glutaric acid methyl esters can further include the polypeptide with esterase active, and enter one Step generation glutaric acid.
Generating such recombinant host of glutaryl-[acp] can further include the polypeptide with thioesterase activity, and And generation glutaric acid.
Such recombinant host of generation glutaryl-CoA can further be included following one or more：I () has sulphur The polypeptide of esterase active, (ii) has the polypeptide of reversible CoA ligase activity, and (iii) has many of CoA- transferase actives Peptide or (iv) have the polypeptide of acylated dehydrogenase activity, and (v) has aldehyde dehydrogenase activity, such as 7- oxo-heptanoic acids dehydrogenase, 6- The polypeptide of oxo caproic acid dehydrogenase or 5- oxopentanoic acid dehydrogenase activities, and further generate glutaric acid or 5- oxopentanoic acids.
The recombinant host of generation 5- oxopentanoic acids or glutaric acid can further be included following one or more：I () has The polypeptide of ω-transaminase activity or (ii) have the polypeptide of carboxylate reductase activity, and further generate 5- aminovaleric acids.
The recombinant host for generating glutaric acid methyl esters can further be included following one or more：I () has ω-transaminase The polypeptide and (iii) that the polypeptide or (ii) of activity have carboxylate reductase activity have the polypeptide of esterase active and further give birth to Into 5- aminovaleric acids.
The recombinant host of generation 5- oxopentanoic acids or glutaric acid can further be included following one or more：I () has The polypeptide of alcohol dehydrogenase activity or (ii) have the polypeptide of carboxylate reductase activity and further generate 5- hydroxypentanoic acids.
The recombinant host for generating glutaric acid methyl esters can further be included following one or more：I () has alcohol dehydrogenase The polypeptide of activity, (ii) has the polypeptide of polypeptide or (iii) with carboxylate reductase activity of esterase active and further gives birth to Into 5- hydroxypentanoic acids.
The recombinant host for generating 5- hydroxypentanoic acids can be included further following one or more：I () has carboxylase also The polypeptide of original enzyme activity and (ii) have the polypeptide of alcohol dehydrogenase activity, and the host further generates 1,5-PD.
The recombinant host for generating 5- hydroxypentanoic acids can be included further following one or more：I () reduces with carboxylic acid The polypeptide of enzymatic activity, the polypeptide and (iii) that (ii) has ω-transaminase activity for one or more has many of alcohol dehydrogenase activity Peptide, the host further generates cadaverine.
The recombinant host for generating 5- aminovaleric acids can be included further following one or more：I () reduces with carboxylic acid The polypeptide of enzymatic activity, and (ii) has the polypeptide of ω-transaminase activity, and the host further generates cadaverine.
The recombinant host for generating 5- oxopentanoic acids can be included further following one or more：I () reduces with carboxylic acid The polypeptide of enzymatic activity and (ii) have the polypeptide of ω-transaminase activity for one or more, and the host further generates cadaverine
The recombinant host for generating 1,5- pentanediols further can have alcohol dehydrogenase activity one or more comprising (i) Polypeptide and (ii) have the polypeptide of ω-transaminase activity for one or more, and the host further generates cadaverine.
The recombinant host for generating 5- aminovaleric acids can be included further following one or more：I () has N- acetyl group The polypeptide of transferase active, (ii) have carboxylate reductase activity polypeptide, (iii) have ω-transaminase activity polypeptide and (iv) polypeptide of deacetylase activity, the host further generates cadaverine.
In engineering approach, enzyme can come from single source, i.e., from a kind of species or category, or can come from various Source, i.e. different plant species or category.The nucleic acid of enzyme described herein is encoded from the identification of various organisms, and can in the public It is easily available in database (such as GenBank or EMBL).
Can be used for one or more described herein any enzyme of C5 building blocks of generation can be with corresponding wild-type enzyme Amino acid sequence have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%th, 93%, 94%, 95%, 97%, 98%, 99% or 100%).It should be appreciated that (such as can be removed based on maturase Any signal sequence) or sequence identity is determined based on immature enzyme (such as including any signal sequence).It is also understood that Initial methionine residue may or may not be present herein on any enzyme sequence of description.
For example, it is described herein with heptanedioyl-polypeptide of [acp] methyl ester methyl esterase active can be with Escherichia coli (referring to Genbank accession number AAC76437.1, SEQ ID NO:1) heptanedioyl-[acp] methyl ester methyl esterase has at least 70% Sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Referring to Fig. 1-3.
For example, the polypeptide with carboxylate reductase activity described herein can be with Mycobacterium marinum (referring to Genbank Accession number ACC40567.1, SEQ ID NO:2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Segniliparus rugosus are (referring to Genbank accession number EFV11917.1, SEQ ID NO:4), shame dirt branch Bacillus is (referring to Genbank accession number ABK75684.1, SEQ ID NO:5), Marseille mycobacteria is (referring to Genbank accession number EIV11143.1,SEQ ID NO:6), or Segniliparus rotundus (referring to Genbank accession number ADG98140.1, SEQ ID NO:7) amino acid sequence of carboxylate reductase has at least 70% sequence identity (homology) (for example, at least 75%th, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).Ginseng See Fig. 6, Fig. 8 and Fig. 9.
For example, the polypeptide with ω-transaminase activity described herein (is logged in blue or green chromabacterium biolaceum referring to Genbank Number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa is (referring to Genbank accession number AAG08191.1, SEQ ID NO: 9), pseudomonas syringae is (referring to Genbank accession number AAY39893.1, SEQ ID NO:10), Rhodobacter (referring to Genbank accession number ABA81135.1, SEQ ID NO:11), Escherichia coli (referring to Genbank accession number AAA57874.1, SEQ ID NO:, or vibrio fluvialis is (referring to Genbank accession number AEA39183.1, SEQ ID NO 12):13) ω-transaminase Amino acid sequence can have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Some in these ω-transaminases are Diamines ω-transaminase.Referring to Fig. 5-7.
For example, the polypeptide with Phosphopantetheinyl transferase activity described herein can be with withered grass gemma Bacillus Phosphopantetheinyl transferase is (referring to Genbank accession number CAA44858.1, SEQ ID NO:Or promise card 14) The Phosphopantetheinyl transferases of Salmonella species NRRL 5646 are (referring to Genbank accession number ABI83656.1, SEQ ID NO:15) amino acid sequence have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Referring to Fig. 4 and Fig. 9.
For example, the polypeptide with esterase active described herein can be with the amino acid sequence of Pseudomonas fluorescens esterase (referring to Genbank accession number AAC60471.2, SEQ ID NO:16) with least 70% sequence identity (homology) (for example At least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Referring to Fig. 4,5,8.
For example, the polypeptide with thioesterase activity described herein can be with Lactobacillus brevis acyl group-[acp] thioesterase (referring to Genbank accession number ABJ63754.1, SEQ ID NO:17), Lactobacillus plantarum acyl group-[acp] thioesterase (referring to Genbank accession number ABJ63754.1, SEQ ID NO:, or Escherichia coli thioesterase is (referring to Genbank accession number 18) AAB59067.1 or AAA24665.1, SEQ ID NO:Amino acid sequence 22-23) has at least 70% sequence identity (same Source property) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Referring to Fig. 4.
For example, it is described herein with malonyl-polypeptide of [acp] O- methyl transferase activities can be with wax-like bud Spore bacillus malonyl-[acp] O- transmethylases is (referring to Genbank accession number AAC76437.1, SEQ ID NO:21) Amino acid sequence have at least 70% sequence identity (homology) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Referring to Fig. 1-3.
For example, the polypeptide with enoyl-CoA reductase activity described herein can be with treponema denticola (ginseng See Genbank accession number AAS11092.1, SEQ ID NO:Or Euglena gracilis are (referring to Genbank accession number 19) AAW66853.1,SEQ ID NO:20) amino acid sequence of enoyl-CoA reductase has at least 70% sequence identity (same Source property) (for example, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).Referring to Fig. 1-3.
The percentage identity (homology) between two kinds of amino acid sequences can as follows be determined.First, using from containing BLAST 2Sequences (Bl2seq) program aligned amino acid sequence of the standalone version BLASTZ of BLASTP 2.0.14 editions.This Standalone version BLASTZ can be available from the website (such as www.fr.com/blast/) of Fish＆Richardson or state of U.S. government Vertical Biotechnology Information center website (www.ncbi.nlm.nih.gov).Explain the directions for use for how using Bl2seq programs May refer to the readme file of BLASTZ.Bl2seq implements the ratio between two kinds of amino acid sequences using BLASTP algorithms Compared with.In order to compare two kinds of amino acid sequences, the following option that Bl2seq is set：- i is set to contain the first amino acid to be compared File (such as C of sequence:\seq1.txt)；- j is set to file (such as C containing the second amino acid sequence to be compared:\ seq2.txt)；- p is set to blastp；- o is set to any desired file name (such as C:\output.txt)；And institute There are other options to remain its default setting.It is, for example possible to use being produced with issuing orders containing between two kinds of amino acid sequences The output file for comparing：C:\Bl2seq–i c:\seq1.txt–j c:\seq2.txt–p blastp–o c:\ output.txt.If two kinds of shared homologys (homogeneity) of comparative sequences, then it is same that the output file specified can be presented those Yuan Xing areas are used as aligned sequences.If two kinds of comparative sequences do not share homology (homogeneity), then the output file specified is not Aligned sequences can be presented.Similar code can be followed nucleotide sequence, blastn is simply used.
Once comparing, matching is determined by the number for calculating the position that same amino acid residue is presented in both sequences Number., divided by the length of full-length polypeptide amino acid sequence, the numerical value of gained then is multiplied by into 100 to determine by with matching number Percentage identity (homology).Notice that percentage identity (homology) value is rounded up to nearest tenths.Example Such as, 78.1 are rounded up to 78.11,78.12,78.13 and 78.14 downwards, and 78.15,78.16,78.17,78.18 and 78.19 are rounded up to 78.2 upwards.It is also noted that length value can always integer.
It will be appreciated that many nucleic acid can encode the polypeptide with specific amino acid sequence.The degeneracy of genetic code is It is as known in the art；I.e. for many amino acid, there are more than a kind of nucleotide triplet for serving as amino acid codes. For example, the codon during the coded sequence of given enzyme can be modified, so that in obtaining particular species (such as bacterium or fungi) Optimum expression, this is carried out using the codon preference table for being suitable for the species.
The functional fragment of any enzyme described herein can also be used in the method for presents.As used herein , term " functional fragment " refers to at least 25% (for example, at least 30%；40%；50%；60%；70%；75%；80%； 85%；90%；95%；98%；99%；100%；Or corresponding maturation, total length, wild-type protein even greater than 100%) The fragments of peptides of the protein of activity.Functional fragment is general but not can be always to be made up of the continuum of protein, wherein the area With functional activity.
This file additionally provides functional variant thereof and (ii) the above-described work(for the enzyme used in the method for (i) presents The functional variant thereof of energy property fragment.Relative to corresponding wild-type sequence, the functional variant thereof of enzyme and functional fragment can contain There are addition, missing or replace.Enzyme with substitution can typically have no more than 50 (such as no more than 1,2,3,4,5,6,7,8, 9th, 10,12,15,20,25,30,35,40 or 50) place's 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor (such as conservative replacement).This is applied to described herein Any enzyme and functional fragment.Conservative replacement is the another kind for having similar features with a kind of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.Conservative replacement Including the substitution in following group：Valine, alanine and glycine；Leucine, valine and isoleucine；Aspartic acid and Glutamic acid；Asparagine and glutamine；Serine, cysteine and threonine；Lysine and arginine；And phenylalanine And tyrosine.Nonpolar hydrophobic acidic amino acid include alanine, leucine, isoleucine, valine, proline, phenylalanine, Tryptophan and methionine.Polar neutral amino acid includes glycine, serine, threonine, cysteine, tyrosine, asparagus fern Acid amides and glutamine.Positively charged (alkalescence) amino acid includes arginine, lysine and histidine.Negatively charged (acid Property) amino acid include aspartic acid and glutamic acid.A kind of member of polarity mentioned above, alkalescence or acid group is by identical group Any substitution of another member can be considered as conservative replacement.Comparatively, non-conservative substitutions is that have with a kind of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor There is the another kind of different characteristic.
Deletion mutants can lack 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 The section (there are two or more amino acid) of individual amino acid or discrete single amino acid.Addition (addition variant) includes Fusion protein, it contains：(a) any enzyme described herein or its fragment；(b) is internal or end (C or N) is unrelated or heterologous Amino acid sequence.In the background of such fusion protein, term " heterologous amino acid sequence " refers to the amino acid sequences different from (a) Row.Heterologous sequence can be for example for purification of recombinant proteins sequence (such as FLAG, polyhistidine (such as six histidines), Agglutinin (HA), glutathione-S-transferase (GST) or maltose-binding protein (MBP)).Heterologous sequence can also be available Make the protein of detectable mark, such as luciferase, green fluorescent protein (GFP) or chloramphenicol acetyltransferase (CAT).In some embodiments, fusion protein contains the signal sequence from another protein.In some host cells In (such as yeast host cell), can be via the expression and/or secretion using Heterologous signal sequences raising target protein.At some In embodiment, fusion protein can contain can be used for for example trigger immune response with generate antibody carrier (such as KLH) or ER or golgiosome stick signal.Heterologous sequence can be different length, and can be in some cases than with it is heterologous The longer sequence of total length target protein of sequence attachment.
Engineering host can naturally express in the enzyme of approach described herein none or some it is (such as a kind of or many Kind, two or more, three or more, four kinds or more plant, five kinds or more plant or six kinds or more plant).In this way, Approach in engineering host can include all exogenous enzymes, or can be comprising both endogenous and exogenous enzymes.Can also destroy The endogenous gene of host is engineered to prevent the formation of undesired metabolin or prevent via other for working intermediate The loss of such intermediate in the approach that enzyme causes.Engineering host is properly termed as recombinant host or recombinant host cell.Such as this Described in text, recombinant host can have as described in this article comprising one or more nucleic acid of polypeptide of coding, the polypeptide Reductase, deacetylase, N- acetyltransferases, malonyl-[acp] O- transmethylases, esterase, thioesterase, water Synthase, dehydrogenase, or ω-transaminase, CoA ligase, the activity of CoA transferases.
Further, it is possible to use the enzyme of separation described herein, uses lysate (such as cell from host microorganism Lysate) as enzyme source, or use the various lysates from different hosts microorganism to carry out one in vitro as enzyme source The generation of kind or various C5 building blocks.
The enzyme of terminal carboxyl groups is produced in the biosynthesis of C5 building blocks
As described in Fig. 4, it is possible to use the i) polypeptide with thioesterase activity, (ii) there is reversible CoA ligase to live Property polypeptide, (iii) has polypeptide of CoA transferase actives, and (iv) has polypeptide of acylated dehydrogenase activity, or (v) has Aldehyde dehydrogenase activity, such as 7- oxo-heptanoic acids dehydrogenase, 6- oxo caproic acid dehydrogenase activities, or 5- oxopentanoic acid dehydrogenase activities Polypeptide, or (vi) have esterase active polypeptide enzymatic formed terminal carboxyl groups.
In some embodiments, by the thioesterase classified under EC 3.1.2.-, such as YciA (SEQ ID NO: 22), tesB (Genbank accession number AAA24665.1, SEQ ID NO:23) or Acot13 (see, for example, Cantu et al., Protein Science,2010,19,1281–1295；Zhuang et al.,Biochemistry,2008,47(9),2789– 2796；Or Naggert et al., J.Biol.Chem., 1991,266 (17), 11044-11050) gene outcome enzymatic shape Into the terminal carboxyl groups for causing glutaric acid to synthesize.
In some embodiments, by CoA- transferases such as glutaconate CoA- transferases (for example, in EC 2.8.3.12 lower classification, such as from acidaminococcus fermentans (Acidaminococcus fermentans)) enzymatic forms and leads Cause the second end carboxylic group of glutaric acid synthesis.See, for example, Buckel et al., 1981, Eur.J.Biochem., 118:315–321.Fig. 4.
In some embodiments, by reversible CoA- ligases such as Succinate-CoA ligase (for example, in EC 6.2.1.5 lower classification, such as from Thermococcus kodakaraensis)) enzymatic forms cause glutaric acid to synthesize the Two terminal carboxyl groups.See, for example, Shikata et al., 2007, J.Biol.Chem., 282 (37):26963–26970.
In some embodiments, by acyl group-[acp] thioesterase classified in EC 3.1.2.-, such as from short breast Bacillus (GenBank accession number ABJ63754.1, SEQ ID NO:4) or from Lactobacillus plantarum (GenBank accession number CCC78182.1,SEQ ID NO:5) acyl group-[acp] thioesterase enzymatic forms the second end carboxyl for causing glutaric acid to synthesize Group.There is such acyl group-[acp] thioesterase C6-C8 chain lengths specificity (see, for example, Jing et al., 2011, BMC Biochemistry,12(44)).See, for example, Fig. 4.
In some embodiments, by aldehyde dehydrogenase, for example, under EC 1.2.1.3 classify aldehyde dehydrogenase (referring to Guerrillot＆Vandecasteele, Eur.J.Biochem., 1977,81,185-192) enzymatic formed cause glutaric acid to close Into second end carboxylic group.Referring to Fig. 4.
In some embodiments, by the aldehyde dehydrogenase classified under EC 1.2.1.-, such as glutaric acid semialdehyde dehydrogenation Enzyme (for example, classifying under EC 1.2.1.20), butanedioic acid-semialdehyde dehydrogenase is (for example, in EC1.2.1.16 or EC 1.2.1.79 Lower classification), or the second end carboxylic group that the aldehyde dehydrogenase enzymatic formation classified under EC 1.2.1.3 causes glutaric acid to synthesize. For example, the aldehyde dehydrogenase classified under EC 1.2.1.- can be the gene outcome of 5- oxopentanoic acids dehydrogenase such as CpnE, 6- oxygen For caproic acid dehydrogenase (such as the gene outcome of the ChnE from Acinetobacter species (Acinetobacter sp.)), or 7- Oxo-heptanoic acid dehydrogenase (for example, gene outcome of the ThnG from Sphingomonas macrogolitabida) (Iwaki et al.,Appl.Environ.Microbiol.,1999,65(11),5158–5162；López-Sánchez et al., Appl.Environ.Microbiol.,2010,76(1),110-118).For example, 6- oxo caproic acid dehydrogenases can be in EC 1.2.1.63 lower classification, the gene outcome of such as ChnE.For example, 7- oxo-heptanoic acids dehydrogenase can divide under EC 1.2.1.- Class.
In some embodiments, by the polypeptide with esterase active such as in EC 3.1.1.-, such as EC 3.1.1.1 the esterase enzymatic or under EC 3.1.1.6 classified forms the second end carboxylic group for causing glutaric acid to synthesize.
The enzyme of terminal amine group is generated in the biosynthesis of C5 building blocks
As described in Fig. 5-7, it is possible to use ω-transaminase or deacetylase enzymatic form terminal amine group.
In some embodiments, (such as classify under EC 2.6.1.48, such as obtain by 5- aminovaleric acids transaminase From Clostridium viride) form first terminal carboxyl groups.Reversible 5- from Clostridium viride Aminovaleric acid transaminase has shown that similar activity (the Barker et for 6-aminocaprolc acid to be converted into adipic acid semialdehyde al.,J.Biol.Chem.,1987,262(19),8994–9003)。
In some embodiments, can by ω-transaminase (such as in EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, classify under EC 2.6.1.48, or EC 2.6.1.82, such as available from blue or green chromabacterium biolaceum (Genbank accession number AAQ59697.1,SEQ ID NO:8), pseudomonas aeruginosa (Genbank accession number AAG08191.1, SEQ ID NO:9), fourth Fragrant pseudomonad (Genbank accession number AAY39893.1, SEQ ID NO:10), Rhodobacter (Genbank accession number ABA81135.1,SEQ ID NO:11), Escherichia coli (Genbank accession number AEA39183.1, SEQ ID NO:12), river Vibrios (Genbank accession number AAA57874.1, SEQ ID NO:, or streptomyces griseus (Streptomyces 13) Griseus)) enzymatic forms a terminal amine group, causes the synthesis of 5- aminopentanols or 5- amino valerals.For example Some the ω-transaminases classified under EC2.6.1.29 or EC 2.6.1.82 are diamines ω-transaminase (such as SEQ ID NO: 11).Referring to Fig. 5 and Fig. 6.
Reversible ω-transaminase (Genbank accession number AAQ59697.1, SEQ ID NO from blue or green chromabacterium biolaceum:8) Through showing similar activity, it receives 6-aminocaprolc acid as amino group donor, and first terminal amine is formed in adipic acid semialdehyde Group (Kaulmann et al., Enzyme and Microbial Technology, 2007,41,628-637).
Reversible 4-Aminobutanoicacid from streptomyces griseus:A-KG transaminase is had shown that for by 6- amino Caproic acid is converted into similar activity (Yonaha the et al., Eur.J.Biochem., 1985,146,101- of adipic acid semialdehyde 106)。
In some embodiments, second terminal amido for causing cadaverine to synthesize is formed by diamine aminotransferase enzymatic Group.For example, can by diamine aminotransferase (under such as EC 2.6.1.29 classification or under such as EC2.6.1.82 classify, it is all Gene outcome (Genbank accession number AAA57874.1, SEQ ID NO of the Tathagata from the YgjG of Escherichia coli:12)) enzymatic is formed Second terminal amine group.
The gene outcome of ygjG receives large quantities of diamines carbon chain lengths substrates, such as putrescine, cadaverine and spermidine (Samsonova et al.,BMC Microbiology,2003,3:2)。
Diamine aminotransferase from coli strain B have been proven that for 1,5 1,5-DAPs activity (Kim, The Journal of Chemistry,1964,239(3),783–786)。
In some embodiments, the acyl group-bad ammonia classified such as under such as EC 3.5.1.17 by deacetylase Sour deacylase, or the acetyl group putrescine deacetylase enzymatic classified such as under such as EC 3.5.1.62 forms second end End amine groups, cause the synthesis of cadaverine.Acetyl group putrescine from micrococcus luteus (Micrococcus luteus) K-11 takes off Acetyl group enzyme receives extensive carbon chain lengths substrate, such as acetyl group putrescine, acetyl group cadaverine and N8- acetyl spermidines (ginseng See such as Suzuki et al., 1986, BBA-General Subjects, 882 (1):140–142).See, Fig. 7.
The enzyme of terminal hydroxyl group is generated in the biosynthesis of C5 building blocks
As described in Fig. 8 and 9, it is possible to use with alcohol dehydrogenase activity such as 6 hydroxycaproic acid dehydrogenase activity, 5- hydroxyls The polypeptide enzymatic of base valeric acid dehydrogenase activity or 4 hydroxybutyric acid dehydrogenase activity forms terminal hydroxyl group.
For example, can be by dehydrogenase, for example, the dehydrogenase classified under EC 1.1.1.-, such as 6 hydroxycaproic acid take off Hydrogen enzyme, for example, classify (such as gene from ChnD) under EC 1.1.1.258,5- hydroxypentanoic acid dehydrogenases, for example, in EC 1.1.1.- lower classification, such as CpnD gene outcome (see, for example, Iwaki et al., 2002, Appl.Environ.Microbiol.,68(11):5671-5684), the 5- hydroxypentanoic acids from Clostridium viride Dehydrogenase, or 4 hydroxybutyric acid dehydrogenase such as gabD (participate in for example, L ü tke-Eversloh＆Steinb ü chel, 1999, FEMS Microbiology Letters,181(1):63-71) enzymatic is formed causes the terminal hydroxyl group of 5- hydroxypentanoic acids. Referring to Fig. 8.
Can be by with EC 1.1.1.- (such as EC 1.1.1.1,1.1.1.2,1.1.1.21, or 1.1.1.184) The polypeptide enzymatic of the alcohol dehydrogenase activity of classification forms terminal hydroxyl group, and it causes the synthesis of 1,5 pentanediols.Referring to Fig. 9.
Bio-chemical pathway
From malonyl-[acp] or malonyl-CoA to glutaric acid methyl esters, glutaryl-CoA or glutaryl- The approach of [acp]
As shown in Fig. 1, can as follows from malonyl-[acp] synthesizing glutaric acid methyl esters：By malonyl-CoA O- transmethylases, classify under such as EC 2.1.1.197, and the gene outcome of such as bioC converts malonyl-[acp] It is malonyl-[acp] methyl esters；Then it is condensed and β -one fatty acyl group-[acp] synthase by with malonyl-[acp], in example Such as EC 2.3.1.-, such as EC 2.3.1.41, classification under EC 2.3.1.179 or EC 2.3.1.180 (such as fabB, fabF or The gene outcome of fabH) it is converted into 3- oxos glutaryl-[acp] methyl esters；Then by 3- hydroxyl acyl-CoA dehydrogenases, For example under EC 1.1.1.-, such as EC 1.1.1.100 classification (gene outcome of such as fabG) is converted into 3- hydroxyls-glutaryl Base-[acp] methyl esters；Then by 3- hydroxyls acyl group-[acp] dehydratase, classify under such as EC 4.2.1.59, such as fabZ Gene outcome be converted into 2,3- dehydrogenations glutaryl-[acp] methyl esters；Then by trans -2- enoyl-CoA reductases, For example under EC 1.3.1.- such as EC 1.3.1.10 classify, the gene outcome of such as fabI is converted into glutaryl-[acp] first Ester；Then (i) is classified by thioesterase under such as EC 3.1.2.-, such as tesB (SEQ ID NO:23), YciA (SEQ ID NO:22) or Acot13, bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) fatty acyl-acp thioesterase (GenBank accession number AAO77182) or Lactobacillus plantarum acyl group-CoA thioesterases (GenBank accession number CCC78182.1) turn Glutaric acid methyl esters or (ii) are turned to by heptanedioyl-[acp] methyl ester methyl esterase, is classified under such as EC 3.1.1.85, such as bioH(SEQ ID NO:1) it is converted into glutaryl-[acp].
As shown in Fig. 2, can as follows from malonyl-CoA synthesizing glutaric acid methyl esters：By malonyl-CoA O- Transmethylase, classifies under such as EC 2.1.1.197, and malonyl-CoA is converted into third by the gene outcome of such as bioC Diacyl-CoA methyl esters；Then by beta-Ketothiolase, classify under such as EC 2.3.1.16, the gene outcome of such as bktB It is condensed with acetyl-CoA or by β -one fatty acyl group-[acp] synthase, is classified under such as EC 2.3.1.180, such as The gene outcome of fabH is converted into 3- oxo glutaryl-CoA methyl esters with malonyl-CoA condensations；Then 3- hydroxyl acyls are passed through Base-CoA dehydrogenases, in such as EC 1.1.1.- such as EC 1.1.1.100 (such as the gene outcome of fabG) or EC 1.1.1.36 classification is converted into 3- hydroxyls-glutaryl-CoA methyl esters under (such as the gene outcome of phaB)；Then alkene acyl is passed through Base-CoA hydrases, classify under such as EC 4.2.1.119, the gene outcome of such as phaJ (Shen et al., Appl.Environ.Microbiol.,2011,77(9),2905–2915；Fukui et al.,Journal of Bacteriology, 1998,180 (3), 667-673) it is converted into 2,3- dehydrogenation glutaryl-CoA methyl esters；Then by it is trans- 2- enoyl-CoA reductases, in such as EC 1.3.1.- such as EC 1.3.1.38, EC1.3.1.8, EC 1.3.1.10 or EC 1.3.1.44 classification is descended to be converted into glutaryl-CoA methyl esters；Then (i) is divided by thioesterase under such as EC 3.1.2.- Class, such as tesB (SEQ ID NO:23), YciA (SEQ ID NO:22) or Acot13, bacteroides thetaiotaomicron fatty acyl-acp thioesterase (GenBank accession number AAO77182) or Lactobacillus plantarum fatty acyl-acp thioesterase (GenBank accession number CCC78182.1) turn Glutaric acid methyl esters or (ii) are turned to by heptanedioyl-[acp] methyl ester methyl esterase, is classified under such as EC 3.1.1.85, such as bioH(SEQ ID NO:1) it is converted into glutaryl-CoA.
As shown in Fig. 3, can as follows from malonyl-CoA synthesizing glutaric acid methyl esters：By malonyl-CoA O- Transmethylase, classifies under such as EC 2.1.1.197, and malonyl-CoA is converted into third by the gene outcome of such as bioC Diacyl-CoA methyl esters；Then by beta-Ketothiolase, classify under such as EC 2.3.1.16, the gene outcome of such as bktB It is condensed with acetyl-CoA or by β -one fatty acyl group-[acp] synthase, is classified under such as EC 2.3.1.180, such as The gene outcome of fabH is converted into 3- oxo glutaryl-CoA methyl esters with malonyl-CoA condensations；Then 3- hydroxyl acyls are passed through Base-CoA dehydrogenases, under such as EC 1.1.1.-, such as EC 1.1.1.35 or EC 1.1.1.157 classification (such as fadB or The gene outcome of hbd) it is converted into 3- hydroxyls-glutaryl-CoA methyl esters；Then by enoyl--CoA hydrases, in such as EC 4.2.1.17 lower classification, the gene outcome of such as crt is converted into 2,3- dehydrogenation glutaryl-CoA methyl esters；Then by it is trans- 2- enoyl-CoA reductases, classify under such as EC 1.3.1.44, and the gene outcome of such as ter or tdter is converted into penta 2 Acyl group-CoA methyl esters；Then (i) is classified by thioesterase under such as EC 3.1.2.-, such as tesB (SEQ ID NO:23), YciA(SEQ ID NO:22) or Acot13, bacteroides thetaiotaomicron fatty acyl-acp thioesterase (GenBank accession number AAO77182) or Lactobacillus plantarum acyl group-CoA thioesterases (GenBank accession number CCC78182.1) is converted into glutaric acid methyl esters or (ii) passes through heptan Two acyls-[acp] methyl ester methyl esterase, classify under such as EC 3.1.1.85, such as bioH (SEQ ID NO:1) it is converted into penta Diacyl-CoA.
Using glutaric acid methyl esters, glutaryl-[acp] or glutaryl-CoA is used as center precursor to glutaric acid or 5- oxygen For the approach of valeric acid
As described in Fig. 4, can by esterase, in such as EC 3.1.1.-, such as EC 3.1.1.1 or EC3.1.1.6, Such as estC (SEQ ID NO:16) is classified under and glutaric acid methyl esters is converted into glutaric acid.
As described in Fig. 4, glutaryl-CoA can be converted into by glutaric acid by the following：(i) thioesterase, in example As classified under EC 3.1.2.-, such as tesB (SEQ ID NO:23), YciA (SEQ ID NO:22) or Acot13, multiform intends bar (GenBank is logged in for bacterium fatty acyl-acp thioesterase (GenBank accession number AAO77182) or Lactobacillus plantarum acyl group-CoA thioesterases Number CCC78182.1) (ii) reversible CoA ligase, classify under such as EC 6.2.1.5, (iii) CoA transferases, for example Classify under EC 2.8.3.- such as EC 2.8.3.12, or (iv) is acylated dehydrogenase, in such as EC 1.2.1.10 or EC 1.2.1.76 lower classification, is such as encoded and aldehyde dehydrogenase by PduB or PduP, is classified under EC 1.2.1.-, such as glutaric acid half Aldehyde dehydrogenase, classifies under such as EC 1.2.1.20, succinic semialdehyde dehydrogenase, in such as EC 1.2.1.16 or EC 1.2.1.79 lower classification, or the aldehyde dehydrogenase classified under EC 1.2.1.3.It is, for example possible to use 5- oxopentanoic acid dehydrogenases, all Such as the gene outcome of CpnE, the gene outcome of 6- oxo caproic acid dehydrogenases such as ChnE, or 7- oxo-heptanoic acid dehydrogenases (for example come From the gene outcome of the ThnG of Sphingomonas macrogolitabida) 5- oxopentanoic acids are converted into glutaric acid.
As described in Fig. 4, can be classified under such as EC 3.1.2.- by thioesterase, such as tesB (SEQ ID NO: 23), YciA (SEQ ID NO:22) or Acot13, bacteroides thetaiotaomicron fatty acyl-acp thioesterase (GenBank accession number ) or Lactobacillus plantarum acyl group-CoA thioesterases (GenBank accession number CCC78182.1) is by glutaryl-[acp] AAO77182 It is converted into glutaric acid.
Using 5- oxopentanoic acids, glutaric acid as center precursor to 5- aminovaleric acids approach
In some embodiments, as follows from center precursor glutaric acid methyl esters synthesis 5- aminovaleric acids：Reduced by carboxylic acid Enzyme, classifies, such as from Mycobacterium marinum (referring to Genbank accession number ACC40567.1, SEQ under such as EC 1.2.99.6 ID NO:2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Marseille mycobacteria (referring to Genbank accession number EIV11143.1, SEQ ID NO:, or Segniliparus rotundus are (referring to Genbank 6) Accession number ADG98140.1, SEQ ID NO:7), with phosphopantetheine transferase reinforcing agent (such as by from withered grass bud Sfp (Genbank accession number CAA44858.1, the SEQ ID NO of spore bacillus:14) gene or the npt from Nocardia (Genbank accession number ABI83656.1, SEQ ID NO:15) gene code) or GriC and GriD gene outcome (Suzuki Et al., J.Antibiot., 2007,60 (6), 380-387) combination glutaric acid methyl esters is converted into glutaric acid semialdehyde methyl esters； Then by ω-transaminase, in such as EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48 classify or under EC 2.6.1.82, such as from blue or green chromabacterium biolaceum (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa is (referring to Genbank accession number AAG08191.1, SEQ ID NO:9), cloves vacation unit cell Bacterium is (referring to Genbank accession number AAY39893.1, SEQ ID NO:10), Rhodobacter (Rhodobacter Sphaeroides) (referring to Genbank accession number ABA81135.1, SEQ ID NO:11), Escherichia coli (step on referring to Genbank Record AAA57874.1, SEQ ID NO:, or vibrio fluvialis (Vibrio fluvialis) is (referring to Genbank accession number 12) AEA39183.1,SEQ ID NO:13) glutaric acid semialdehyde methyl esters is converted into 5- aminopentanoic acid methyl esters；Then by EC 3.1.1.- the esterase of classification under, the acetyl group classified under the Carboxylesterase classified under such as EC 3.1.1.1 or EC 3.1.1.6 Esterase is converted into 5- aminovaleric acids.For example, esterase can be the gene outcome of estC.Referring to Fig. 5.
In some embodiments, as follows from center precursor glutaric acid methyl esters synthesis 5- aminovaleric acids：By EC 3.1.1.- the Carboxylesterase or EC classified under esterase (such as the gene outcome of estC) such as EC 3.1.1.1 of lower classification 3.1.1.6 glutaric acid methyl esters is converted into glutaric acid by the acetyl esterase of lower classification；Then by carboxylate reductase, in such as EC 1.2.99.6 lower classification, the gene outcome of such as car, with phosphopantetheine transferase reinforcing agent (such as by from withered Sfp (Genbank accession number CAA44858.1, the SEQ ID NO of careless bacillus:14) gene or from Nocardia Npt (Genbank accession number ABI83656.1, SEQ ID NO:15) gene code) or GriC and GriD gene outcome Glutaric acid is converted into 5- oxopentanoic acids by the combination of (Suzuki et al., J.Antibiot., 2007,60 (6), 380-387)； Then ω-transaminase (such as EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, EC are passed through 2.6.1.82 such as SEQ ID NOs:11) 8,10 be converted into 5- aminovaleric acids.Referring to Fig. 5.
In some embodiments, as follows from center precursor glutaric acid methyl esters synthesis 5- aminovaleric acids：Reduced by carboxylic acid Enzyme, classifies, such as from Mycobacterium marinum (referring to Genbank accession number ACC40567.1, SEQ under such as EC 1.2.99.6 ID NO:2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Marseille mycobacteria (referring to Genbank accession number EIV11143.1, SEQ ID NO:, or Segniliparus rotundus are (referring to Genbank 6) Accession number ADG98140.1, SEQ ID NO:7), with phosphopantetheine transferase reinforcing agent (such as by from withered grass bud Sfp (Genbank accession number CAA44858.1, the SEQ ID NO of spore bacillus:14) gene or the npt from Nocardia (Genbank accession number ABI83656.1, SEQ ID NO:15) gene code) or GriC and GriD gene outcome (Suzuki Et al., J.Antibiot., 2007,60 (6), 380-387) combination glutaric acid methyl esters is converted into glutaric acid semialdehyde methyl esters； Then by the carboxyl of classification under esterase (such as the gene outcome of estC) such as EC 3.1.1.1 of classification under EC 3.1.1.- Glutaric acid semialdehyde methyl esters is converted into 5- oxopentanoic acids by the acetyl esterase classified under esterase or EC 3.1.1.6；Then pass through ω-transaminase, in such as EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or Classify under EC 2.6.1.82, such as from blue or green chromabacterium biolaceum (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8) Or pseudomonas syringae is (referring to Genbank accession number AAY39893.1, SEQ ID NO:10) 5- aminovaleric acids are converted into.Referring to Fig. 5.
Using glutaric acid methyl esters as center precursor to 5- hydroxypentanoic acids approach
As described in Fig. 8, can as follows from center precursor glutaric acid methyl esters synthesis 5- hydroxypentanoic acids：By EC 3.1.1.- Under the Carboxylesterase or EC 3.1.1.6 classified under esterase (such as the gene outcome of estC) such as EC 3.1.1.1 of lower classification Glutaric acid methyl esters is converted into glutaric acid by the acetyl esterase of classification；Then by carboxylate reductase, in such as EC 1.2.99.6 Lower classification, the gene outcome of such as car, with phosphopantetheine transferase reinforcing agent (such as by from bacillus subtilis Sfp (Genbank accession number CAA44858.1, the SEQ ID NO of bacterium:14) gene or the npt from Nocardia (Genbank accession number ABI83656.1, SEQ ID NO:15) gene code) or GriC and GriD gene outcome (Suzuki Et al., J.Antibiot., 2007,60 (6), 380-387) combination glutaric acid is converted into glutaric acid semialdehyde；Then pass through Dehydrogenase, classifies under such as EC 1.1.1.-, such as 6 hydroxycaproic acid dehydrogenase, (example of classifying under such as EC1.1.1.258 Such as gene from ChnD), 5- hydroxypentanoic acid dehydrogenases are classified under such as EC 1.1.1.-, the gene outcome of such as CpnD (see, for example, Iwaki et al., 2002, Appl.Environ.Microbiol., 68 (11):, or 4- hydroxyls 5671-5684) Butyryl dehydrogenase such as gabD be converted into 5- hydroxypentanoic acids (see, for example, L ü tke-Eversloh＆Steinb ü chel, 1999, FEMS Microbiology Letters,181(1):63–71).Referring to Fig. 7.
As described in Fig. 8, can as follows from center precursor glutaric acid methyl esters synthesis 5- hydroxypentanoic acids：Reduced by carboxylic acid Enzyme, classifies, such as from Mycobacterium marinum (referring to Genbank accession number ACC40567.1, SEQ under such as EC 1.2.99.6 ID NO:2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Marseille mycobacteria (referring to Genbank accession number EIV11143.1, SEQ ID NO:, or Segniliparus rotundus are (referring to Genbank 6) Accession number ADG98140.1, SEQ ID NO:7), with phosphopantetheine transferase reinforcing agent (such as by from withered grass bud Sfp (Genbank accession number CAA44858.1, the SEQ ID NO of spore bacillus:14) gene or the npt from Nocardia (Genbank accession number ABI83656.1, SEQ ID NO:15) gene code) or GriC and GriD gene outcome (Suzuki Et al., J.Antibiot., 2007,60 (6), 380-387) combination glutaric acid methyl esters is converted into glutaric acid semialdehyde methyl esters； Then by the carboxyl of classification under esterase (such as the gene outcome of estC) such as EC 3.1.1.1 of classification under EC 3.1.1.- The acetyl esterase classified under esterase or EC 3.1.1.6 is converted into glutaric acid semialdehyde；Then by dehydrogenase, in such as EC 1.1.1.- lower classification, such as 6 hydroxycaproic acid dehydrogenase, classification (such as base from ChnD under such as EC 1.1.1.258 Cause), 5- hydroxypentanoic acid dehydrogenases are classified under such as EC 1.1.1.-, the gene outcome of such as CpnD, or 4 hydroxybutyric acid Dehydrogenase, such as gabD are converted into 5- hydroxypentanoic acids.
As described in Fig. 8, can as follows from center precursor glutaric acid methyl esters synthesis 5- hydroxypentanoic acids：Carboxylate reductase, For example under EC 1.2.99.6 classify, such as from Mycobacterium marinum (referring to Genbank accession number ACC40567.1, SEQ ID NO:2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Marseille mycobacteria (referring to Genbank accession number EIV11143.1, SEQ ID NO:6), or Segniliparus rotundus (referring to Genbank log in Number ADG98140.1, SEQ ID NO:7), with phosphopantetheine transferase reinforcing agent (such as by from bacillus subtilis Sfp (Genbank accession number CAA44858.1, the SEQ ID NO of bacterium:14) gene or the npt from Nocardia (Genbank accession number ABI83656.1, SEQ ID NO:15) gene code) or GriC and GriD gene outcome (Suzuki Et al., J.Antibiot., 2007,60 (6), 380-387) combination glutaric acid methyl esters is converted into glutaric acid semialdehyde methyl esters； Then by alcohol dehydrogenase, in such as EC 1.1.1.- (such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21 or EC 1.1.1.184 classify under), such as YMR318C, the gene outcome of YqhD, or the egg with GenBank accession number CAA81612.1 White matter is converted into 5- hydroxyl methyls；Then the esterase (gene outcome of such as estC) by classifying under EC 3.1.1.- is all Acetyl esterase as classified under the Carboxylesterase or EC 3.1.1.6 of classification under EC 3.1.1.1 is converted into 5- hydroxypentanoic acids.
Using 5- aminovaleric acids, 5- hydroxypentanoic acids, or glutaric acid semialdehyde are used as the approach of center precursor to cadaverine
As described in Fig. 4, as follows from center precursor 5- aminovaleric acid synthesis cadaverines：By carboxylate reductase, in such as EC 1.2.99.6 lower classification, the gene outcome and phosphopantetheine transferase reinforcing agent of such as car are (such as by from withered grass The sfp genes of bacillus or the npt gene codes from Nocardia) or GriC from streptomyces griseus and GriD 5- aminovaleric acids are converted into 5- amino valerals by the combination of gene outcome；Then by ω-transaminase, in such as EC 2.6.1.-, such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or classify under EC 2.6.1.82, Such as from blue or green chromabacterium biolaceum (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa (referring to Genbank accession number AAG08191.1, SEQ ID NO:9), pseudomonas syringae is (referring to Genbank accession number AAY39893.1,SEQ ID NO:, or Escherichia coli are (referring to Genbank accession number AAA57874.1, SEQ ID NO 10):12) 5- amino valerals are converted into cadaverine.
The carboxylate reductase and reinforcing agent npt or sfp encoded by the gene outcome of car have extensive substrate specificity, Including end bi-functional C4 and C5 carboxylic acid (Venkitasubramanian et al., Enzyme and Microbial Technology,2008,42,130–137)。
In some embodiments, synthesize from center precursor 5- hydroxypentanoic acids (it can be with generation as shown in Figure 8) as follows Cadaverine：By carboxylate reductase, classify under such as EC 1.2.99.6, such as (stepped on referring to Genbank from Mycobacterium marinum Record ACC40567.1, SEQ ID NO:2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Segniliparus rugosus are (referring to Genbank accession number EFV11917.1, SEQ ID NO:4), Mycobacterium massiliense are (referring to Genbank accession number EIV11143.1, SEQ ID NO:6), or Segniliparus rotundus are (referring to Genbank accession number ADG98140.1, SEQ ID NO:7), with phosphopan tetheine sulfydryl Ethamine transferase reinforcing agent is (such as by sfp (Genbank accession number CAA44858.1, the SEQ ID from bacillus subtilis NO:14) gene or npt (Genbank accession number ABI83656.1, the SEQ ID NO from Nocardia:15) gene is compiled Code), or the gene outcome (Suzuki et al., J.Antibiot., 2007,60 (6), 380-387) of GriC＆GriD combination 5- hydroxypentanoic acids are converted into 5- hydrogenation of hydroxypentylaldehyd；Then by ω-transaminase, in such as EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or classify under EC 2.6.1.82, such as from blue or green chromabacterium biolaceum (ginseng See Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa is (referring to Genbank accession number AAG08191.1,SEQ ID NO:9), pseudomonas syringae is (referring to Genbank accession number AAY39893.1, SEQ ID NO: 10), Rhodobacter is (referring to Genbank accession number ABA81135.1, SEQ ID NO:11), Escherichia coli are (referring to Genbank Accession number AAA57874.1, SEQ ID NO:, or vibrio fluvialis is (referring to Genbank accession number AEA39183.1, SEQ ID 12) NO:13) 5- oxo amylalcohols are converted into 5- aminopentanols；Then by alcohol dehydrogenase, in such as EC 1.1.1.- (such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) under classify, such as YMR318C (Genbank log in Number CAA90836.1) or YqhD (coming from Escherichia coli, GenBank accession number AAA69178.1) gene outcome (Liu et al.,Microbiology,2009,155,2078–2085；Larroy et al.,2002,Biochem J.,361(Pt 1), 163–172；Jarboe, 2011, Appl.Microbiol.Biotechnol., 89 (2), 249-257) or stepped on Genbank The protein of record CAA81612.1 is converted into 5- amino valerals；Then it is all in such as EC 2.6.1.- by ω-transaminase Classify such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or under EC 2.6.1.82, such as from green grass or young crops Chromabacterium biolaceum is (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa (steps on referring to Genbank Record AAG08191.1, SEQ ID NO:9), pseudomonas syringae is (referring to Genbank accession number AAY39893.1, SEQ ID NO:, or Escherichia coli are (referring to Genbank accession number AAA57874.1, SEQ ID NO 10):12) it is converted into cadaverine.Referring to figure 6。
In some embodiments, as follows from center precursor 5- aminovaleric acid synthesis cadaverines：By N- acetyltransferases Such as lysine N- acetyltransferases, classify under such as EC 2.3.1.32 by 5- aminovaleric acids be converted into N5- acetyl group- 5- aminovaleric acids；Then by the gene outcome and phosphopantetheine transferase reinforcing agent of carboxylate reductase such as car (such as by the sfp genes from bacillus subtilis or the npt gene codes from Nocardia), or from grey strepto- The combination of the gene outcome of the GriC and GriD of bacterium is converted into N5- acetyl group -5- amino valerals；Then by ω-transaminase, Such as EC 2.6.1.-, such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 Lower classification, such as from pseudomonas aeruginosa (referring to Genbank accession number AAG08191.1, SEQ ID NO:9), cloves is false single Born of the same parents bacterium is (referring to Genbank accession number AAY39893.1, SEQ ID NO:10), Rhodobacter is (referring to Genbank accession number ABA81135.1,SEQ ID NO:11), Escherichia coli are (referring to Genbank accession number AAA57874.1, SEQ ID NO:12), Or vibrio fluvialis is (referring to Genbank accession number AEA39183.1, SEQ ID NO:13) N5- acetyl group -1,5- diaminos are converted into Base pentane；Then by acetyl group putrescine deacetylase (classifying under such as EC 3.5.1.17 or EC 3.5.1.62) conversion It is cadaverine.Referring to Fig. 7
In some embodiments, as follows from center precursor glutaric acid semialdehyde synthesis cadaverine：By carboxylate reductase, in example As classified under EC 1.2.99.6, such as from Segniliparus rotundus (referring to Genbank accession number ADG98140.1,SEQ ID NO:7), with phosphopantetheine transferase reinforcing agent (such as by from bacillus subtilis Sfp (Genbank accession number CAA44858.1, SEQ ID NO:21) gene or the npt (Genbank from Nocardia Accession number ABI83656.1, SEQ ID NO:22) gene code), or GriC＆GriD gene outcome (Suzuki et al., J.Antibiot., 2007,60 (6), 380-387) combination glutaric acid semialdehyde is converted into glutaraldehyde；Then by ω-turn ammonia Enzyme, in such as EC2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 lower classification, is converted into 5- amino valerals；Then by ω-transaminase, in such as EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or classify under EC 2.6.1.82, such as from livid purple Color bacillus is (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa (logs in referring to Genbank Number AAG08191.1, SEQ ID NO:9), pseudomonas syringae is (referring to Genbank accession number AAY39893.1, SEQ ID NO: , or Escherichia coli are (referring to Genbank accession number AAA57874.1, SEQ ID NO 10):12) it is converted into cadaverine.Referring to Fig. 7.
In some embodiments, as follows from center precursor 1,5-PD synthesis cadaverine：By alcohol dehydrogenase, for example Classify under EC 1.1.1.- (such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184), such as YMR318C (Genbank accession number CAA90836.1) or YqhD (coming from Escherichia coli, GenBank accession number AAA69178.1) Gene outcome (Liu et al., Microbiology, 2009,155,2078-2085；Larroy et al.,2002, Biochem J.,361(Pt 1),163–172；Jarboe,2011,Appl.Microbiol.Biotechnol.,89(2), 249-257) or 1,5- pentanediols are converted into 5- hydrogenation of hydroxypentylaldehyd by the protein with Genbank accession number CAA81612.1；Connect By ω-transaminase, in such as EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, classify or under EC 2.6.1.82, such as from blue or green chromabacterium biolaceum (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa is (referring to Genbank accession number AAG08191.1, SEQ ID NO:9), cloves vacation unit cell Bacterium is (referring to Genbank accession number AAY39893.1, SEQ ID NO:10), Rhodobacter is (referring to Genbank accession number ABA81135.1,SEQ ID NO:11), Escherichia coli are (referring to Genbank accession number AAA57874.1, SEQ ID NO:12), Or vibrio fluvialis is (referring to Genbank accession number AEA39183.1, SEQ ID NO:13) 5- oxo valerals are converted into 5- amino Amylalcohol；Then by alcohol dehydrogenase, such as EC 1.1.1.- (such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, Or EC 1.1.1.184) under classify, such as YMR318C (Genbank accession number CAA90836.1) or YqhD (come from large intestine bar Bacterium, GenBank accession number AAA69178.1) gene outcome (Liu et al., Microbiology, 2009,155,2078- 2085；Larroy et al.,2002,Biochem J.,361(Pt 1),163–172；Jarboe,2011, Appl.Microbiol.Biotechnol., 89 (2), 249-257) or albumen with Genbank accession number CAA81612.1 Matter is converted into 5- amino valerals；Then by ω-transaminase, in such as EC 2.6.1.-, such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or under EC 2.6.1.82 classify, such as from blue or green chromabacterium biolaceum (referring to Genbank accession number AAQ59697.1, SEQ ID NO:8), pseudomonas aeruginosa is (referring to Genbank accession number AAG08191.1,SEQ ID NO:9), pseudomonas syringae is (referring to Genbank accession number AAY39893.1, SEQ ID NO: , or Escherichia coli are (referring to Genbank accession number AAA57874.1, SEQ ID NO 10):12) it is converted into cadaverine.Referring to Fig. 6.
Using 5- hydroxypentanoic acids as center precursor to 1,5- pentanediols approach
As described in Fig. 9, from center, precursor 5- hydroxypentanoic acids synthesize 1,5 pentanediols as follows：By carboxylate reductase, in example As classified under EC 1.2.99.6, such as from Mycobacterium marinum (referring to Genbank accession number ACC40567.1, SEQ ID NO: 2), mycobacterium smegmatis is (referring to Genbank accession number ABK71854.1, SEQ ID NO:3), Segniliparus rugosus (referring to Genbank accession number EFV11917.1, SEQ ID NO:4), Marseille mycobacteria is (referring to Genbank accession number EIV11143.1,SEQ ID NO:6), or Segniliparus rotundus (referring to Genbank accession number ADG98140.1, SEQ ID NO:7), with phosphopantetheine transferase reinforcing agent (such as by the sfp from bacillus subtilis (Genbank accession number CAA44858.1, SEQ ID NO:14) gene or the npt (Genbank accession numbers from Nocardia ABI83656.1,SEQ ID NO:15) gene code), or GriC＆GriD gene outcome (Suzuki et al., J.Antibiot., 2007,60 (6), 380-387) combination 5- hydroxypentanoic acids are converted into 5- hydrogenation of hydroxypentylaldehyd；Then alcohol is passed through Dehydrogenase, in such as EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) Lower classification, such as YMR318C (Genbank accession number CAA90836.1) or YqhD (come from Escherichia coli, GenBank accession number AAA69178.1 gene outcome) (see, for example, Liu et al., Microbiology, 2009,155,2078-2085； Larroy et al.,2002,Biochem J.,361(Pt 1),163–172；Or Jarboe, 2011, Appl.Microbiol.Biotechnol., 89 (2), 249-257) or albumen with Genbank accession number CAA81612.1 Be converted into for 5- hydrogenation of hydroxypentylaldehyd by matter (coming from Geobacillus stearothermophilus (Geobacillus stearothermophilus)) 1,5 pentanediols.Referring to Fig. 9.
Training strategy
In some embodiments, training strategy needs to realize aerobic, anaerobism, micro- aerobic or mixing oxygen/denitrification culture Condition.Vitro characterization needs to maintain the micro- aerobic training strategy of extremely low dissolved oxygen concentration (to see, for example, for the enzyme of oxygen sensitivity Chayabatra&Lu-Kwang,Appl.Environ.Microbiol.,2000,66(2),493 0 498；Wilson and Bouwer,1997,Journal of Industrial Microbiology and Biotechnology,18(2-3),116- 130)。
In some embodiments, Cyclic culture strategy needs realizing anaerobic culture conditions and realizing aerobic condition of culture Between alternately.
In some embodiments, training strategy needs nutrition to limit, such as nitrogen, and phosphate or oxygen are limited.
In some embodiments, it is possible to use the final electron acceptor such as nitrate in addition to oxygen.In some implementations In scheme, can use realized using the cell retention strategy of such as ceramic membrane and maintain fed-batch or continuously ferment period High-cell density.
In some embodiments, the primary carbon source to feeding medium during fermentation in one or more C5 building blocks synthesis can be with source From biological or abiotic raw material.
In some embodiments, the biological raw material can be or can be from monose, disaccharides, lignocellulosic, half Cellulose, cellulose, lignin, levulic acid and formic acid, triglycerides, glycerine, aliphatic acid, agricultural wastes, concentration vinasse (condensed distillers'solubles) or municipal waste.
Several microorganisms (such as Escherichia coli, hookworm corrupt bacteria, Pseudomonas oleovorans, pseudomonas putida and Yarrowialipolytica) in demonstrate the crude glycerol from production of biodiesel effective catabolism (Lee et al., Appl.Biochem.Biotechnol.,2012,166:1801–1813；Yang et al.,Biotechnology for Biofuels,2012,5:13；Meijnen et al.,Appl.Microbiol.Biotechnol.,2011,90:885- 893)。
Demonstrated via precursor propionyl in several organisms (such as hookworm corrupt bacteria and pseudomonas putida) To effective catabolism (Jaremko of levulic acid derived from lignocellulosic in the synthesis of the 3- hydroxypentanoic acids of base-CoA And Yu, 2011, see above；Martin and Prather,J.Biotechnol.,2009,139:61–67).
Fragrance derived from lignin is demonstrated in several microorganisms (such as pseudomonas putida, hookworm corrupt bacteria) Effective catabolism (Bugg et al., Current Opinion in of compounds of group such as benzoic acid analog Biotechnology,2011,22,394–400；Pérez-Pantoja et al.,FEMS Microbiol.Rev.,2008, 32,736–794)。
Agricultural wastes are demonstrated in several microorganisms (including Yarrowialipolytica), and (such as olive mill gives up Water) effective utilization (Papanikolaou et al., Bioresour.Technol., 2008,99 (7):2419-2428).
It has been directed to several microorganisms (such as Escherichia coli, corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis Bacterium) fermentable saccharide is demonstrated (such as from cellulose, hemicellulose, sugarcane and beet molasses, cassava, corn and other agricultures Industry source monose and disaccharides) effective utilization (see, e.g. Hermann et al, J.Biotechnol., 2003,104: 155–172；Wee et al.,Food Technol.Biotechnol.,2006,44(2):163–172；Ohashi et al., J.Bioscience and Bioengineering,1999,87(5):647-654)。
Effective utilization (Li of the furfural from various agricultural lignocellulosic source has been demonstrated for hookworm corrupt bacteria et al.,Biodegradation,2011,22:1215–1225).
In some embodiments, the abiotic raw material can be or can be derived from natural gas, synthesis gas, CO2/H2, first Alcohol, ethanol, benzoate, non-volatile residue (NVR) or the alkali wash water (caustic from cyclohexane oxidation process Wash) waste stream or terephthalic acid (TPA)/isophathalic acid mixture waste stream.
It has been directed to effective catabolism that methylotrophic yeast pichia pastoris phaff demonstrates methyl alcohol.
For clostridium kluyveri demonstrate ethanol effective catabolism (Seedorf et al., Proc.Natl.Acad.Sci.USA,2008,105(6)2128-2133).
CO is demonstrated for hookworm corrupt bacteria2And H2(it can be derived from natural gas and other chemistry and petrochemistry comes Source) effective catabolism (Prybylski et al., Energy, Sustainability and Society, 2012,2: 11)。
It has been directed to effective decomposition that multiple-microorganism (such as Young clostridium and from producing and ethanol clostridium) demonstrates synthesis gas Metabolism ( et al.,Applied and Environmental Microbiology,2011,77(15):5467– 5475)。
Multiple-microorganism (such as acidophilic bacteria and hookworm corrupt bacteria) has been directed to demonstrate from hexamethylene mistake Effective catabolism (Ramsay et al., Applied and of the non-volatile residue waste stream of journey Environmental Microbiology,1986,52(1):152–156)。
In some embodiments, the host microorganism is prokaryotes.For example, the prokaryotes can be thin Bacterium, from Escherichia (Escherichia) such as Escherichia coli (Escherichia coli)；From fusobacterium (Clostridia) such as Young clostridium (Clostridium ljungdahlii), from producing and ethanol clostridium (Clostridium ) or clostridium kluyveri (Clostridium kluyveri) autoethanogenum；From corynebacterium (Corynebacteria) such as corynebacterium glutamicum (Corynebacterium glutamicum)；From greedy copper Pseudomonas (Cupriavidus) such as hookworm corrupt bacteria (Cupriavidus necator) or the greedy copper bacterium (Cupriavidus of resistance to metal metallidurans)；From pseudomonas (Pseudomonas) such as Pseudomonas fluorescens (Pseudomonas Fluorescens), pseudomonas putida (Pseudomonas putida) or Pseudomonas oleovorans (Pseudomonas oleavorans)；From Delftiatsuruhatensis category (Delftia), such as acidophilic bacteria (Delftia acidovorans)； From bacillus (Bacillus) such as Bacillus subtillis (Bacillus subtillis)；From lactobacillus (Lactobacillus) such as Lactobacillus delbrueckii (Lactobacillus delbrueckii)；Or from lactococcus (Lactococcus) such as Lactococcus lactis (Lactococcus lactis).The prokaryotes can also be the source of gene with Building specifically described herein can generate one or more recombinant host cell of C5 building blocks.
In some embodiments, host microorganism is eucaryote.For example, eucaryote can be with filamentous fungi, such as From aspergillus (Aspergillus) such as aspergillus niger (Aspergillus niger).Or, eucaryote can be yeast, example Such as come from saccharomyces (Saccharomyces) such as saccharomyces cerevisiae (Saccharomyces cerevisiae)；From complete Chi Shi ferment Category (Pichia) such as pichia pastoris phaff (Pichia pastoris)；From Ye Luoweiya saccharomyces (Yarrowia) as solved Fat Ye Luoweiya yeast (Yarrowia lipolytica)；From Issatchenkia (Issatchenkia) such as her Sa ferment of east Female (Issathenkia orientalis)；From Debaryomyces (Debaryomyces) such as the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii)；From Arxula category such as Arxula adenoinivorans；Or tie up ferment from Crewe Female Pseudomonas (Kluyveromyces) such as lactic acid yeast kluyveromyces (Kluyveromyces lactis).The eucaryote may be used also Specifically described herein one or more recombinant host of C5 building blocks can be generated building to be the source of gene.
Metabolic engineering
Presents provides method, and methods described is related to the step of being less than all steps for all above-mentioned approach descriptions. This method can relate to 1,2,3,4,5,6,7,8,9,10,11,12 or more in for example such step.In this method In the case of all or fewer than step, first, and step unique in some embodiments can be in listed step Any step.
Additionally, recombinant host described herein can include any combinations in above-mentioned enzyme so that in the step One or more, such as in such step 1,2,3,4,5,6,7,8,9,10 or more can be real in recombinant host Apply.Presents provides listed and genetically engineered one or more (examples to express any enzyme enumerated in presents Such as, 2,3,4,5,6,7,8,9,10,11,12 or more kinds of) any category and the host cell of kind of recombinant forms.In this way, example Such as, host cell can contain exogenous nucleic acid, one or more step of its coding catalysis any approach described herein Enzyme.
In addition, presents is recognized, in the case of the substrate for receiving CoA activation is had described as in enzyme, exist with The related similar enzymatic activity of [acp] bound substrates, the enzyme class that it is not necessarily to the same.
Additionally, presents is recognized, in the case where enzyme has described as (the R)-enantiomter for receiving substrate, deposit In the similar enzymatic activity related to (the S)-enantiomter of substrate, the enzyme class that it is not necessarily to the same.
Presents be also to be recognized that have shown that enzyme receive specific co-factor such as NADPH or cosubstrate such as acetyl group- In the case of CoA, many enzymes are general places being catalyzed in specific enzymatic activity in terms of a large amount of difference co-factors or cosubstrate is received (promiscuous) of main property.Additionally, presents is recognized, have to for example specific confactor such as NADH in enzyme high special Property in the case of, have to confactor NADPH high specific with it is similar or it is identical activity enzyme can be different enzymes Species.
In some embodiments, the enzyme in approach outlined herein is via non-immediate or reasonable enzyme method for designing Enzyme engineering result, it is therefore intended that improve activity, improve specificity, reduce feedback inhibition, reduction prevent, improves enzyme dissolving Degree, change stereospecificity, or change confactor specificity.
In some embodiments, in the approach that can will be herein summarized via additive type or chromosomal integration method Enzyme gene puts into (gene dose) to (that is, overexpression) in the organism of the genetic modification of gained.
In some embodiments, it is possible to use genome rank (genome-scale) systems biology technology such as flux Equilibrium analysis (Flux Balance Analysis) is designed for guiding carbon flow to the genome rank of C5 building blocks Weaken or knock out strategy.
Weaken strategy include but is not limited to using transposons, homologous recombination (dual crossing method), mutagenesis, enzyme inhibitor and RNAi is disturbed.
In some embodiments, it is possible to use flux group (fluxomic), metabolome (metabolomic) and transcription Thing group (transcriptomal) data come the systems biology technology informing or support genome rank, thus by carbon flow Amount designs the decrease of genome rank or knocks out strategy during being oriented to C5 building blocks.
In some embodiments, host microorganism can be improved to highly concentrated via the continuous culture in selective environment The tolerance of the C5 building blocks of degree.
In some embodiments, weaken or the endogenous biological chemical network of lifting host microorganism ensures acetyl with (1) The intracellular availability of base-CoA and malonyl-CoA, (2) create NADH or NADPH imbalances, and it can be via one or more C5 building blocks formation balance, (3) prevent cause and the central metabolites thing comprising C5 building blocks, the degraded of center precursor and/ Or (4) ensure the effective outflow from cell.
Need the intracellular availability of acetyl-CoA with synthesize C5 building blocks some embodiments in, can be in host Weaken the endogenous enzymes of the hydrolysis of catalysis acetyl-CoA, such as short chain thioesterase in organism.
Need the condensation of acetyl-CoA and malonyl-CoA with synthesize C5 building blocks some embodiments in, can It is the endogenous beta-Ketothiolase of acetoacetyl-CoA, such as AtoB to weaken one or more catalysis only acetyl-CoA condensation Or the endogenous gene products of phaA.
Need the intracellular availability of acetyl-CoA with synthesize C5 building blocks some embodiments in, product can be weakened The endogenous phosphoric acid acetic acid transferase of raw acetic acid, such as pta (Shen et al., Appl.Environ.Microbiol., 2011, 77(9):2905–2915)。
Need the intracellular availability of acetyl-CoA with synthesize C5 building blocks some embodiments in, second can be weakened The endogenous gene of the encoding acetate kinase in sour route of synthesis, such as ack.
Need the intracellular availability of acetyl-CoA and NADH with synthesize C5 building blocks some embodiments in, can be with Weaken catalysis degradation of pyruvate into the endogenous gene of the enzyme of lactic acid, lactic dehydrogenase (the Shen et for such as being encoded by ldhA Al., 2011, see above).
Need the intracellular availability of acetyl-CoA and NADH with synthesize C5 building blocks some embodiments in, can be with Weaken the enzyme that PEP (phophoenolpyruvate) is degraded into fumaric acid for coding catalysis, such as The endogenous gene of menaquinol- fumaric acid oxidoreducing enzyme, and such as frdBC (see, for example, Shen et al., 2011, see Above).
Need the intracellular availability of acetyl-CoA and NADH with synthesize C5 building blocks some embodiments in, can be with Weaken the enzyme that acetyl-CoA is degraded into ethanol for coding catalysis, the endogenous gene of the alcohol dehydrogenase for such as being encoded by adhE (Shen et al., 2011, see above).
In some embodiments, in the case where approach needs excessive NADH co-factors to synthesize C5 building blocks, can With the overexpression restructuring formate dehydrogenase gene (Shen et al., 2011, see above) in host organisms.
In some embodiments, can in host organisms overexpression acetyl-CoA carboxylase.
In some embodiments, can overexpression 3-phosphoglyceric acid dehydroenase, 3- phosphoserine ammonia in host Circulated as SAMe using producing serine one or more in based transferase and phosphoserine phosphatase Methyl donor.
In some embodiments, can in host overexpression methanol dehydrogenase or formaldehyde dehydrogenase allowing via first The Methanol Decomposition metabolism of acid.
In some embodiments, NADH the or NADPH co-factors of excess are needed in approach to synthesize the feelings of C5 building blocks Under condition, the unbalanced transhydrogenase of dissipation co-factor can be weakened.
In some embodiments, it is the enzyme of ethanol that can weaken coding catalysis by degradation of pyruvate, and such as pyruvic acid takes off The endogenous gene of carboxylic acid.
In some embodiments, the enzyme that coding catalyzing iso-butane alcohol is produced can be weakened, such as 2- keto acid decarboxylases is interior Source gene.
Need the intracellular availability of acetyl-CoA with synthesize C5 building blocks some embodiments in, can be in micro- life Gene outcome (Satoh et al., the J.Bioscience and of overexpression restructuring acetyl-CoA synzyme such as acs in thing Bioengineering,2003,95(4):335–341)。
In some embodiments, can be by weakening endogenous glucose -6- phosphoric acid isomerases (EC 5.3.1.9) by carbon Flow is directed in pentose phosphate cycle to improve the supply of NADPH.
In some embodiments, can be by overexpression 6-phosphogluconate dehydrogenase and/or transketolase by carbon Flow is redirected in pentose phosphate cycle to improve supply (the Lee et al., 2003, Biotechnology of NADPH Progress,19(5),1444–1449)。
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized Under, can in host organisms overexpression gene such as encode pyridine nucleotide transhydrogenase UdhA (Brigham et al., Advanced Biofuels and Bioproducts,2012,Chapter 39,1065-1090)。
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized Under, GAPDH gene such as GapN (Brigham et can be recombinated by overexpression in host organisms Al., 2012, see above).
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized In, malate dehydrogenase (malate dehydrogenase) gene such as maeA or maeB (Brigham can be recombinated by overexpression in host organisms Et al., 2012, see above).
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized Under, can in host organisms overexpression restructuring G 6 PD gene mutations such as zwf (Lim et al., J.Bioscience and Bioengineering,2002,93(6),543-549)。
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized Under, can in host organisms overexpression restructuring fructose 1,6 diphosphatase gene such as fbp (Becker et al., J.Biotechnol.,2007,132:99-109)。
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized Under, endogenous triose-phosphate isomerase (EC 5.3.1.1) can be weakened.
In some embodiments, the situation of excessive NADPH co-factors is needed in approach in C5 building blocks are synthesized Under, can in host organisms overexpression recombinant glucose dehydrogenase such as gdh gene outcome (Satoh et al., J.Bioscience and Bioengineering,2003,95(4):335–341)。
In some embodiments, the endogenous enzymes for promoting NADPH to change into NADH, the conversion such as NADH can be weakened Circulation is produced, it can be via the paddy classified under EC 1.4.1.2 (NADH- specificity) and EC1.4.1.4 (NADPH- specificity) The change of propylhomoserin dehydrogenase is produced.
In some embodiments, the endogenous glutamic acid by the use of both NADH and NADPH as co-factor can be weakened to take off Hydrogen enzyme (EC 1.4.1.3).
In some embodiments, can via as with small soluble protein, such as fusion of maltose-binding protein Protein expression, make the dissolving of molten film combination enoyl-CoA reductase (Gloerich et al., FEBS Letters, 2006, 580,2092–2096)。
In some embodiment party of the host using natural accumulation polyhydroxyalkanoatefrom (polyhydroxyalkanoates) In case, the endogenous polyhydroxyalkanoate synthase of enzyme can be weakened in host strain.
In some embodiments of the host using natural accumulation liposome, weaken the enzyme that coding participates in liposome synthesis Gene.
In some embodiments, can in host overexpression L-alanine dehydrogenase with from acetone acid regeneration the third ammonia of L- The sour amino group donor as ω-transamination reaction.
In some embodiments, can overexpression Pidolidone dehydrogenase, Pidolidone synzyme or paddy in host Propylhomoserin synthase regenerates Pidolidone with from a-KG, used as the amino group donor of ω-transamination reaction.
In some embodiments, the heptanedioyl-CoA dehydrogenases classified under enzyme such as EC 1.3.1.62 can be weakened；Acyl Base-CoA dehydrogenases, for example, classifying under EC 1.3.8.7 or EC 1.3.8.1；And/or glutaryl-CoA dehydrogenases, example Such as, the glutaryl-CoA dehydrogenases classified under EC 1.3.8.6, its degraded causes and the center generation including C5 building blocks Xie Wuhe centers precursor.
In some embodiments, the endogenous enzymes via coacetylase esterification activation C5 building blocks can be weakened, such as, The CoA- ligases (such as glutaryl-CoA synzyme) classified under EC 6.2.1.6.
In some embodiments, can appoint by the genetically engineered structural modification of cell membrane or raising C5 building blocks What association transport protein increased activity amplifies outflow of the C5 building blocks through cell membrane to extracellular culture medium.
Can be by overexpression substrate spectrum multidrug transporter wide, the such as Blt from bacillus subtilis (Woolridge et al.,1997,J.Biol.Chem.,272(14):8864–8866)；AcrB from Escherichia coli and AcrD (Elkins＆Nikaido, 2002, J.Bacteriol., 184 (23), 6490-6499), from staphylococcus aureus NorA (the Ng et al., 1994, Antimicrob Agents Chemother, 38 of (Staphylococcus aereus) (6), 1345-1355), or Bmr (Neyfakh, 1992, Antimicrob Agents from bacillus subtilis Chemother, 36 (2), 484-485) enhancing or amplify cadaverine outflow.
Corynebacterium glutamicum (Corynebacterium can such as be come from by overexpression Solute Transport albumen Glutamicum lysE transport proteins enhancing) or amplify 5- aminovaleric acids and cadaverine outflow (Bellmann et al., 2001,Microbiology,147,1765–1774)。
The enhancing of SucE transport proteins or amplification by overexpression dicarboxylic acids transport protein such as from Corynebacterium glutamicum The outflow (Huhn et al., Appl.Microbiol.＆Biotech., 89 (2), 327-335) of glutaric acid.
C5 building blocks are generated using recombinant host
Generally, can be by providing host microorganism, and with the culture medium containing suitable carbon source described above The microorganism for providing is provided and generates one or more C5 building block.Usually, culture medium and/or culture can cause that microorganism gives birth to Grow to enough density and effectively produce C5 building blocks.For large-scale production process, it is possible to use any method, such as not Place's description (Manual of Industrial Microbiology and Biotechnology, 2nd Edition, Editors:A.L.Demain and J.E.Davies,ASM Press；With Principles of Fermentation Technology,P.F.Stanbury and A.Whitaker,Pergamon).In short, be inoculated with specified microorganisms containing Suitable culture medium big tank (for example, 100 gallons, 200 gallons, 500 gallons, or more tank).After inoculation, micro- life is cultivated Thing with allow produce biomass.Once desired biomass is reached, can be by the media transfer containing microorganism to second Tank.This second tank can be any size.It is less for example, second tank can be larger, or with first tank phase With size.Generally, second tank is more than first so that can be to adding extra culture from first culture medium of tank Base.In addition, culture medium in this second tank can be identical or different with the culture medium that uses in first tank.
Once transfer, can incubate microorganism to allow to generate C5 building blocks.Once generation, it is possible to use any method is come Separate C5 building blocks.For example, can be via adsorption method from zymotic fluid selective recovery C5 building blocks.In glutaric acid and 5- amino In the case of valeric acid, can be crystallized via evaporation and/or crystallisation by cooling via the eluent obtained by evaporation further concentration, And reclaim crystal via centrifugation.In the case of cadaverine and 1,5-PD, can realize that desired product is pure using distillation Degree.The present invention is further described in the examples below, and embodiment does not limit the scope of the present invention described in claims.
Embodiment
Embodiment 1
Using glutaric acid semialdehyde as substrate and the enzymatic activity of the ω-transaminase for forming 5- aminovaleric acids
The nucleotide sequence for encoding N-terminal His labels is added to and is separately encoded SEQ ID NO:8 and 10 ω-transaminase The gene from blue or green chromabacterium biolaceum and Rhodobacter (referring to Figure 10) so that the ω of N-terminal plus HIS labels-turn can be generated Ammonia enzyme.Modified gene that each is obtained is cloned into pET21a expression vectors under the control of T7 promoters, and by each Expression vector is transformed into BL21 [DE3] escherichia coli host.The recombinant escherichia coli strain of gained is containing 50mL LB cultures Cultivated in the case where being shaken with 230rpm in 37 DEG C in the 250mL diastatochromogenes of base and antibiotic selective pressure.Use 1mM IPTG Each culture is induced at 16 DEG C overnight.
Via the granule from each diastatochromogenes for inducing is harvested by centrifugation.Resuspended every kind of granule, and via ultrasound Treatment cracking.Cell fragment and supernatant are separated via centrifugation, and it is neutral i.e. using acellular extraction in enzyme assay method Thing.
By final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM 5- aminovaleric acids, 10mM pyruvic acid and 100 μM Reverse (reverse direction) is carried out in the buffer solution of the phosphate of pyridoxal 5 ' composition, and (i.e. 5- aminovaleric acids are to glutaric acid Semialdehyde) enzyme assay method.By by the cell-free extract of ω-aminotransferase gene product or empty vector control be added to containing Start each enzyme assay reaction in the measure buffer solution of 5- aminovaleric acids, and at 25 DEG C in the case of 250rpm shakes Incubate 4 hours.Quantify to be formed from the ALANINE of pyruvic acid by RP-HPLC.
The control for not having each only enzyme of 5- aminovaleric acids shows that pyruvic acid to the low baseline of ALANINE is converted.Referring to figure 17.The gene outcome of SEQ ID NO 8 receives 5- aminovaleric acids as substrate, such as confirms for empty vector control.Referring to Figure 18.
Confirm the enzymatic activity of positive (i.e. glutaric acid semialdehyde to 5- aminovaleric acids) to the transaminase of SEQ ID NO 10. By final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM glutaric acid semialdehydes, 10mML- alanine and 100 μM of pyridoxals 5 ' Enzyme assay method is carried out in the buffer solution of phosphate composition.By by the nothing of ω-aminotransferase gene product or empty vector control Cell extract is added in the measure buffer solution containing glutaric acid semialdehyde to start each enzyme assay reaction, and at 25 DEG C Incubated 4 hours in the case where 250rpm shakes.Quantify pyruvic acid by RP-HPLC to be formed.
The gene outcome of SEQ ID NO 10 receives glutaric acid semialdehyde as substrate, such as confirms for empty vector control.Ginseng See Figure 19.Confirm the invertibity of ω-transaminase activity, show that SEQ ID NO 8 and the ω-transaminase of SEQ ID NO 10 connect By glutaric acid semialdehyde as substrate, and synthesize 5- aminovaleric acids as product.
Embodiment 2
Using 5- hydroxypentanoic acids as substrate and the enzymatic activity of the carboxylate reductase for forming 5- hydrogenation of hydroxypentylaldehyd
The nucleotide sequence for encoding His- labels is added to and is separately encoded SEQ ID NO:2-4,6, and 7 carboxylic acid reduction Enzyme (respectively GenBank accession number ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, and ADG98140.1) from Mycobacterium marinum, mycobacterium smegmatis, Segniliparus rugosus, Marseille mycobacteria and The gene (referring to Figure 10) of Segniliparus rotundus so that the carboxylate reductase of N-terminal plus HIS labels can be generated.Will The gene cloning of each modification in pET Duet expression vectors, in coding from bacillus subtilis plus His labels phosphorus By the sfp genes of sour pantetheine transferase, both under the control of T7 promoters.With the expression from embodiment 3 Be transformed into every kind of expression vector in BL21 [DE3] escherichia coli host together by carrier.In the case where being shaken with 230rpm, Contain the weights for cultivating every kind of gained in the 250mL flask cultures under 50mL LB culture mediums and antibiotic selective pressure at 37 DEG C Group coli strain.At 37 DEG C every kind of culture is induced using auto-induction culture medium overnight.
Via the granule that the diastatochromogenes from every kind of induction are harvested by centrifugation.Resuspended every kind of granule, and via ultrasound Treatment cracking.Cell fragment and supernatant are separated via centrifugation.Using Ni affinity chromatographys from supernatant purification of carboxylic acids reductase With phosphopantetheine transferase, it is diluted in 50mM HEPES buffer solutions (pH=7.5) with 10 times, and via ultrafiltration Concentration.
By final concentration 50mM HEPES buffer solutions (pH=7.5), 2mM 5- hydrogenation of hydroxypentylaldehyd, 10mM MgCl2, 1mM The triplicate enzymatic activity (i.e. 5- hydroxypentanoic acids to 5- hydrogenation of hydroxypentylaldehyd) that carries out is determined in ATP, and the buffer solution of 1mM NADPH compositions Method.Contain 5- hydroxyls by the way that the carboxylate reductase of purifying and phosphopantetheine transferase or empty vector control are added to The measure buffer solution of valeric acid starts each enzyme assay reaction, and in incubation at room temperature 20 minutes.By the extinction at 340nm The consumption of degree monitoring NADPH.The control for not having each only enzyme of 5- hydroxypentanoic acids shows that the low baseline of NADPH is consumed.Referring to figure 12。
SEQ ID NO:The gene outcome (being strengthened by the gene outcome of sfp) of 2-4,6 and 7 receives 5- hydroxypentanoic acids the bottom of as Thing, such as confirms (referring to Figure 14), and synthesize 5- hydrogenation of hydroxypentylaldehyd for empty vector control.
Embodiment 3
For 5- amino valerals, the enzymatic activity of the ω-transaminase of 5- oxo amylalcohols is formed
The nucleotide sequence for encoding N-terminal His labels is added to and is separately encoded SEQ ID NO:ω-the transaminase of 8-13 Blue or green chromabacterium biolaceum, pseudomonas aeruginosa, pseudomonas syringae, Rhodobacter, Escherichia coli and vibrio fluvialis gene are (referring to figure 10) so that the ω-transaminase of N-terminal plus HIS labels can be generated.Modified gene is cloned under the control of T7 promoters In pET21a expression vectors.Each expression vector is transformed into BL21 [DE3] escherichia coli host.By the restructuring obtained by each Coli strain in the 250mL diastatochromogenes containing 50mL LB culture mediums and antibiotic selective pressure in 37 DEG C with The lower culture of 230rpm shakes.Using 1mM IPTG each culture is induced at 16 DEG C overnight.
Via the granule from each diastatochromogenes for inducing is harvested by centrifugation.Resuspended every kind of granule, and via ultrasound Treatment cracking.Cell fragment and supernatant are separated via centrifugation, and it is neutral i.e. using acellular extraction in enzyme assay method Thing.
By final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM 5- aminopentanols, 10mM pyruvic acid and 100 μM Reversely (i.e. 5- aminopentanols to 5- oxos amylalcohol) enzyme assay method is carried out in the buffer solution of the phosphate of pyridoxal 5 ' composition.It is logical Cross and the cell-free extract of ω-aminotransferase gene product or empty vector control is added to the measure buffering containing 5- aminopentanols Start each enzyme assay reaction in liquid, and incubated 4 hours in the case of 250rpm shakes at 25 DEG C.By RP- HPLC quantifies the formation of ALANINE.
The control for not having each only enzyme of 5- aminopentanols shows that pyruvic acid to the low baseline of ALANINE is converted.Referring to figure 17。
SEQ ID NO:The gene outcome of 8-13 receives 5- aminopentanols as substrate, such as confirms for empty vector control (referring to Figure 13), and synthesize 5- oxo amylalcohols as product.In view of the invertibity of ω-transaminase activity is (referring to implementation Example 1), may infer that SEQ ID NO:The gene outcome of 8-13 receives 5- oxo amylalcohols as substrate, and forms 5- amino penta Alcohol.
Embodiment 4
Using cadaverine as substrate and the enzymatic activity of the ω-transaminase for forming 5- amino valerals
The nucleotide sequence for encoding N-terminal His labels is added to and is separately encoded SEQ ID NO:8-10 and 12 ω-turn ammonia The blue or green chromabacterium biolaceum of enzyme, pseudomonas aeruginosa, pseudomonas syringae, and bacillus coli gene (referring to Figure 10) so that Ke Yisheng Into N-terminal plus the ω-transaminase of HIS labels.Modified gene is cloned into pET21a expression vectors under the control of T7 promoters In.Each expression vector is transformed into BL21 [DE3] escherichia coli host.Recombinant escherichia coli strain obtained by each is existed Trained in the case where being shaken with 230rpm in 37 DEG C in 250mL diastatochromogenes containing 50mL LB culture mediums and antibiotic selective pressure Support.Using 1mM IPTG each culture is induced at 16 DEG C overnight.
Via the granule from each diastatochromogenes for inducing is harvested by centrifugation.Resuspended every kind of granule, and via ultrasound Treatment cracking.Cell fragment and supernatant are separated via centrifugation, and it is neutral i.e. using acellular extraction in enzyme assay method Thing.
By final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM cadaverines, 10mM pyruvic acid and 100 μM of pyridoxals Reversely (i.e. cadaverine to 5- amino valeral) enzyme assay method is carried out in the buffer solution of 5 ' phosphate composition.By by ω-turn ammonia The cell-free extract of enzyme gene product or empty vector control is added in the measure buffer solution containing cadaverine to start each enzyme Determination of activity is reacted, and is incubated 4 hours in the case of 250rpm shakes at 25 DEG C.ALANINE shape is quantified by RP-HPLC Into.
Low baseline of the control with pyruvic acid to ALANINE for not having each only enzyme of cadaverine is converted.Referring to Figure 17.
SEQ ID NO:8-10 and 12 gene outcome receives cadaverine as substrate, such as confirms (ginseng for empty vector control See Figure 11), and synthesize 5- amino valerals as product.In view of the invertibity of ω-transaminase activity is (referring to embodiment 1) SEQ ID NO, be may infer that:8-10 and 12 gene outcome receives 5- amino valerals as substrate, and forms cadaverine.
Embodiment 5
Using N5- acetyl group -1,5- 1,5-DAPs, and form the ω-transaminase of N5- acetyl group -5- amino valerals Enzymatic activity
The SEQ ID NO that N-terminal adds His labels are determined using buffer solution:8,10-13 ω-transaminase (referring to embodiment 3, And Figure 10) be used for N5- acetyl group -1,5- 1,5-DAPs are converted into the activity of N5- acetyl group -5- amino valerals, described slow Fliud flushing is by final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM N5- acetyl group -1,5- 1,5-DAPs, 10mM acetone Acid and 100 μM of phosphate of pyridoxal 5 ' are constituted.By by the cell-free extract of ω-aminotransferase gene product or empty vector control It is added to containing N5- acetyl group -1, each enzyme assay is started in the measure buffer solution of 5- 1,5-DAPs and is reacted, and 25 DEG C incubate 4 hours in the case where 250rpm shakes.Quantify ALANINE by RP-HPLC to be formed.
The control for not having each only enzyme of N5- acetyl group -1,5- 1,5-DAPs shows that pyruvic acid is low to ALANINE Baseline is converted.Referring to Figure 17.
SEQ ID NO:8,10 gene outcome receives N5- acetyl group -1, and 5- 1,5-DAPs are such as directed to sky as substrate Vehicle Control is confirmed (referring to Figure 15), and synthesizes N5- acetyl group -5- amino valerals as product.
In view of the invertibity (referring to embodiment 1) of ω-transaminase activity, SEQ ID NO:8,10 gene outcome receives N5- acetyl group -5- amino valeral forms N5- acetyl group -1,5- 1,5-DAPs as substrate.
Embodiment 6
Using glutaric acid semialdehyde as substrate and the enzymatic activity of the carboxylate reductase for forming glutaraldehyde
Using glutaric acid semialdehyde as substrate determine N-terminal add His labels SEQ ID NO 7 carboxylate reductase (referring to Embodiment 2 and Figure 10).Enzyme assay method is carried out in triplicate using buffer solution, and the buffer solution is by final concentration 50mM HEPES buffer solution (pH=7.5), 2mM glutaric acid semialdehydes, 10mM MgCl2, 1mM ATP and 1mM NADPH compositions.By will be pure The carboxylate reductase and phosphopantetheine transferase or empty vector control of change are added to the measure containing glutaric acid semialdehyde Buffer solution starts enzyme assay reaction, then in incubation at room temperature 20 minutes.Monitor NADPH's by the absorbance at 340nm Consumption.The control for not having the only enzyme of glutaric acid semialdehyde shows that the low baseline of NADPH is consumed.Referring to Figure 12.
The gene outcome (being strengthened by the gene outcome of sfp) of SEQ ID NO 7 receives glutaric acid semialdehyde as substrate, such as pin Empty vector control is confirmed (referring to Figure 16), and synthesis of glutaraldehyde.
Embodiment 7
Using glutaric acid methyl esters as substrate and the enzymatic activity of the carboxylate reductase for forming glutaric acid semialdehyde methyl esters
The carboxylate reductase that N-terminal adds the SEQ ID NO 2-4 and 7 of His labels is determined as substrate using glutaric acid methyl esters (referring to embodiment 2 and Figure 10).Enzyme assay method is carried out in triplicate using buffer solution, and the buffer solution is by final concentration 50mM HEPES buffer solution (pH=7.5), 2mM glutaric acid methyl esters, 10mM MgCl2, 1mM ATP and 1mM NADPH compositions.By will be pure The carboxylate reductase and phosphopantetheine transferase or empty vector control of change are added to the measure containing glutaric acid semialdehyde Buffer solution starts enzyme assay reaction, then in incubation at room temperature 20 minutes.Monitor NADPH's by the absorbance at 340nm Consumption.The control for not having the only enzyme of glutaric acid methyl esters shows that the low baseline of NADPH is consumed.Referring to Figure 12.
The gene outcome (being strengthened by the gene outcome of sfp) of SEQ ID NO 2-4 and 7 receives glutaric acid methyl esters the bottom of as Thing, such as confirms (referring to Figure 20) for empty vector control, and synthesizing glutaric acid semialdehyde methyl esters.
Embodiment 8
Using N5- acetyl group -1,5- 1,5-DAPs, 1- aminopentanes and 1- aminoheptanes are used as glutaric acid semialdehyde methyl esters Substitute (proxy) and formed 5- aminopentanoic acid methyl esters ω-transaminase enzymatic activity.
The SEQ ID NO that N-terminal adds His labels are determined using buffer solution:8,10-13 (referring to embodiments 3, and Figure 10) ω-transaminase is used for N5- acetyl group -1, and 5- 1,5-DAPs are converted into the activity of N5- acetyl group -5- amino valerals, described Buffer solution is by final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM N5- acetyl group -1,5- 1,5-DAPs, 10mM third Ketone acid and 100 μM of phosphate of pyridoxal 5 ' are constituted.It is added to by by the cell-free extract of ω-transaminase or empty vector control Containing N5- acetyl group -1, the measure buffer solution of 5- 1,5-DAPs starts each enzyme assay reaction, then with 250rpm Incubated 4 hours in 25 DEG C in the case of shake.Quantify the formation of ALANINE by RP-HPLC.
The control for not having each only enzyme of N5- acetyl group -1,5- 1,5-DAPs shows that pyruvic acid is low to ALANINE Baseline is converted.Referring to Figure 17.
SEQ ID NO:8,10-13 gene outcome receives N5- acetyl group -1, and 5- 1,5-DAPs are used as substrate, such as pin Empty vector control is confirmed (referring to Figure 15), and synthesizes N5- acetyl group -5- amino valerals as product.
In view of the invertibity (referring to embodiment 1) of ω-transaminase activity, SEQ ID NOs:The gene outcome of 8,10-13 connects By (shielded) primary amine that acetyl group is shielded, such as N5- acetyl group -5- amino valeral as substrate, formed N5- acetyl group - 1,5- 1,5-DAPs.
The SEQ ID NO that N-terminal adds His labels are determined using buffer solution:8,10-13 (referring to embodiments 3, and Figure 10) ω-transaminase is used to be converted into 1- aminopentanes the activity of valeral, and the buffer solution is by final concentration 50mM HEPES buffer solutions (pH=7.5), 10mM 1- aminopentanes, 10mM pyruvic acid and 100 μM of phosphate of pyridoxal 5 ' are constituted.By by ω-transaminase Or the cell-free extract of empty vector control is added to the measure buffer solution containing 1- aminopentanes and starts each enzyme assay Reaction, then incubates 4 hours in the case where being shaken with 250rpm in 25 DEG C.Quantify the formation of ALANINE by RP-HPLC.
The control for not having each only enzyme of 1- aminopentanes shows that pyruvic acid to the low baseline of ALANINE is converted.Referring to figure 17。
SEQ ID NO:The gene outcome of 8-13 receives 1- aminopentanes as substrate, such as confirms for empty vector control (referring to Figure 21), and synthesize valeral as product.
In view of the invertibity (referring to embodiment 1) of ω-transaminase activity, SEQ ID NO:The gene outcome of 8-13 receives 5 Carbon primary amine 1- aminopentanes form valeral as substrate.
The SEQ ID NO that N-terminal adds His labels are determined using buffer solution:8-13 (referring to embodiments 3, and Figure 10) are used for will 1- aminoheptanes are converted into the activity of the ω-transaminase of enanthaldehyde, and the buffer solution is by final concentration 50mM HEPES buffer solutions (pH= 7.5), 10mM 1- aminoheptanes, 10mM pyruvic acid and 100 μM of phosphate of pyridoxal 5 ' are constituted.By by ω-transaminase or sky The cell-free extract of vehicle Control is added to the measure buffer solution containing 1- aminoheptanes and starts each enzyme assay reaction, Then incubated 4 hours in 25 DEG C in the case where being shaken with 250rpm.Quantify the formation of ALANINE by RP-HPLC.
The control for not having each only enzyme of 1- aminoheptanes shows that pyruvic acid to the low baseline of ALANINE is converted.Referring to figure 17。
SEQ ID NO:The gene outcome of 8-13 receives 1- aminoheptanes as substrate, such as confirms for empty vector control (referring to Figure 21), and synthesize enanthaldehyde as product.
In view of the invertibity (referring to embodiment 1) of ω-transaminase activity, SEQ ID NO:The gene outcome of 8-13 receives 7 Carbon primary amine 1- aminoheptanes form enanthaldehyde as substrate.
In view of (representing omega to the N5- acetyl group-1,5- 1,5-DAPs and C5-C7 chain length primary amine of acetyl group shielding Monofunctional substrate) activity that proves, SEQ ID NO:8-13 forms 5- ammonia with glutaric acid semialdehyde methyl esters is received as substrate The high possibility of base methyl valerate.
Embodiment 9
As substrate and heptanedioyl-[acp] methyl esters first of glutaryl-CoA is formed using glutaryl-CoA methyl esters The enzymatic activity of base ester enzyme
The sequence for encoding C-terminal His labels is added to coding SEQ ID NO:1 heptanedioyl-[acp] methyl ester methyl esterase The gene from Escherichia coli (referring to Figure 10) so that heptanedioyl-[acp] methyl ester methyl of C-terminal plus HIS labels can be generated Esterase.The modified gene of gained is cloned into pET28b+ expression vectors under the control of T7 promoters, and expression is carried Body is transformed into BL21 [DE3] escherichia coli host.Recombinant escherichia coli strain obtained by each is being contained into 100mL LB trainings Cultivated in the case where being shaken with 230rpm in 37 DEG C in the 500mL diastatochromogenes of foster base and antibiotic selective pressure.Use 0.3mM IPTG induces each culture overnight at 18 DEG C.
Via the granule from each diastatochromogenes for inducing is harvested by centrifugation.Resuspended every kind of granule, and via ultrasound Treatment cracking.Cell fragment and supernatant are separated via centrifugation.Using Ni- affinity chromatographys heptanedioyl-[acp] is purified from supernatant Methyl ester methyl esterase, carries out buffering fluid exchange, and is arrived in 20mM HEPES buffer solutions (pH=7.5) via being concentrated by ultrafiltration, and And in 4 DEG C of storages.
What is be made up of final concentration 25mM TrisHCl buffer solutions (pH=7.0) and 5 [mM] glutaryl-CoA methyl esters Enter to be about to the enzyme assay method that glutaryl-CoA methyl esters is converted into glutaryl-CoA in triplicate in buffer solution.By inciting somebody to action Heptanedioyl-[acp] methyl ester methyl esterase is added to the measure buffer solution containing glutaryl-CoA methyl esters with final concentration 10 [μM] In come start enzyme assay reaction, and 30 DEG C 250rpm shake in the case of incubate 1 hours.Quantify penta by LC-MS Diacyl-CoA is formed.
There is no the substrate glutaryl-CoA of the control without display trace of the only substrate of enzyme.Referring to Figure 23.SEQ ID The heptanedioyl of NO.1-[acp] methyl ester methyl esterase receives glutaryl-CoA methyl esters as substrate, and synthesize glutaryl- CoA such as confirms as product via LC-MS.Referring to Figure 23.
Other embodiments
It should be understood that, although detailed description describe the present invention with reference to it, but description above be intended to explanation and It is not to limit the scope of the present invention limited by the scope of the appended claims.Other side, advantage and modification are in appended power In the range of sharp claim.
Sequence table
<110>Technology Co., Ltd of English Weida
<120>Method for generating glutaric acid and glutaric acid methyl esters
<130> 35643-0071WO1
<150> US 62/012,586
<151> 2014-06-16
<150> US 62/012,722
<151> 2014-06-06
<160> 23
<170> FastSEQ for Windows Version 4.0
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Met Asn Asn Ile Trp Trp Gln Thr Lys Gly Gln Gly Asn Val His Leu
1 5 10 15
Val Leu Leu His Gly Trp Gly Leu Asn Ala Glu Val Trp Arg Cys Ile
20 25 30
Asp Glu Glu Leu Ser Ser His Phe Thr Leu His Leu Val Asp Leu Pro
35 40 45
Gly Phe Gly Arg Ser Arg Gly Phe Gly Ala Leu Ser Leu Ala Asp Met
50 55 60
Ala Glu Ala Val Leu Gln Gln Ala Pro Asp Lys Ala Ile Trp Leu Gly
65 70 75 80
Trp Ser Leu Gly Gly Leu Val Ala Ser Gln Ile Ala Leu Thr His Pro
85 90 95
Glu Arg Val Gln Ala Leu Val Thr Val Ala Ser Ser Pro Cys Phe Ser
100 105 110
Ala Arg Asp Glu Trp Pro Gly Ile Lys Pro Asp Val Leu Ala Gly Phe
115 120 125
Gln Gln Gln Leu Ser Asp Asp Phe Gln Arg Thr Val Glu Arg Phe Leu
130 135 140
Ala Leu Gln Thr Met Gly Thr Glu Thr Ala Arg Gln Asp Ala Arg Ala
145 150 155 160
Leu Lys Lys Thr Val Leu Ala Leu Pro Met Pro Glu Val Asp Val Leu
165 170 175
Asn Gly Gly Leu Glu Ile Leu Lys Thr Val Asp Leu Arg Gln Pro Leu
180 185 190
Gln Asn Val Ser Met Pro Phe Leu Arg Leu Tyr Gly Tyr Leu Asp Gly
195 200 205
Leu Val Pro Arg Lys Val Val Pro Met Leu Asp Lys Leu Trp Pro His
210 215 220
Ser Glu Ser Tyr Ile Phe Ala Lys Ala Ala His Ala Pro Phe Ile Ser
225 230 235 240
His Pro Ala Glu Phe Cys His Leu Leu Val Ala Leu Lys Gln Arg Val
245 250 255
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<213>Mycobacterium marinum
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Met Ser Pro Ile Thr Arg Glu Glu Arg Leu Glu Arg Arg Ile Gln Asp
1 5 10 15
Leu Tyr Ala Asn Asp Pro Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala
20 25 30
Ile Thr Ala Ala Ile Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile Ile
35 40 45
Glu Thr Val Met Thr Gly Tyr Ala Asp Arg Pro Ala Leu Ala Gln Arg
50 55 60
Ser Val Glu Phe Val Thr Asp Ala Gly Thr Gly His Thr Thr Leu Arg
65 70 75 80
Leu Leu Pro His Phe Glu Thr Ile Ser Tyr Gly Glu Leu Trp Asp Arg
85 90 95
Ile Ser Ala Leu Ala Asp Val Leu Ser Thr Glu Gln Thr Val Lys Pro
100 105 110
Gly Asp Arg Val Cys Leu Leu Gly Phe Asn Ser Val Asp Tyr Ala Thr
115 120 125
Ile Asp Met Thr Leu Ala Arg Leu Gly Ala Val Ala Val Pro Leu Gln
130 135 140
Thr Ser Ala Ala Ile Thr Gln Leu Gln Pro Ile Val Ala Glu Thr Gln
145 150 155 160
Pro Thr Met Ile Ala Ala Ser Val Asp Ala Leu Ala Asp Ala Thr Glu
165 170 175
Leu Ala Leu Ser Gly Gln Thr Ala Thr Arg Val Leu Val Phe Asp His
180 185 190
His Arg Gln Val Asp Ala His Arg Ala Ala Val Glu Ser Ala Arg Glu
195 200 205
Arg Leu Ala Gly Ser Ala Val Val Glu Thr Leu Ala Glu Ala Ile Ala
210 215 220
Arg Gly Asp Val Pro Arg Gly Ala Ser Ala Gly Ser Ala Pro Gly Thr
225 230 235 240
Asp Val Ser Asp Asp Ser Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser
245 250 255
Thr Gly Ala Pro Lys Gly Ala Met Tyr Pro Arg Arg Asn Val Ala Thr
260 265 270
Phe Trp Arg Lys Arg Thr Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile
275 280 285
Thr Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gln Ile Leu
290 295 300
Tyr Gly Thr Leu Cys Asn Gly Gly Thr Ala Tyr Phe Val Ala Lys Ser
305 310 315 320
Asp Leu Ser Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu
325 330 335
Leu Thr Phe Val Pro Arg Val Trp Asp Met Val Phe Asp Glu Phe Gln
340 345 350
Ser Glu Val Asp Arg Arg Leu Val Asp Gly Ala Asp Arg Val Ala Leu
355 360 365
Glu Ala Gln Val Lys Ala Glu Ile Arg Asn Asp Val Leu Gly Gly Arg
370 375 380
Tyr Thr Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Asp Glu Met Lys
385 390 395 400
Ala Trp Val Glu Glu Leu Leu Asp Met His Leu Val Glu Gly Tyr Gly
405 410 415
Ser Thr Glu Ala Gly Met Ile Leu Ile Asp Gly Ala Ile Arg Arg Pro
420 425 430
Ala Val Leu Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe
435 440 445
Leu Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Val Lys Thr Asp
450 455 460
Ser Leu Phe Pro Gly Tyr Tyr Gln Arg Ala Glu Val Thr Ala Asp Val
465 470 475 480
Phe Asp Ala Asp Gly Phe Tyr Arg Thr Gly Asp Ile Met Ala Glu Val
485 490 495
Gly Pro Glu Gln Phe Val Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys
500 505 510
Leu Ser Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe
515 520 525
Gly Asp Ser Pro Leu Val Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala
530 535 540
Arg Ala Tyr Leu Leu Ala Val Ile Val Pro Thr Gln Glu Ala Leu Asp
545 550 555 560
Ala Val Pro Val Glu Glu Leu Lys Ala Arg Leu Gly Asp Ser Leu Gln
565 570 575
Glu Val Ala Lys Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
580 585 590
Phe Ile Ile Glu Thr Thr Pro Trp Thr Leu Glu Asn Gly Leu Leu Thr
595 600 605
Gly Ile Arg Lys Leu Ala Arg Pro Gln Leu Lys Lys His Tyr Gly Glu
610 615 620
Leu Leu Glu Gln Ile Tyr Thr Asp Leu Ala His Gly Gln Ala Asp Glu
625 630 635 640
Leu Arg Ser Leu Arg Gln Ser Gly Ala Asp Ala Pro Val Leu Val Thr
645 650 655
Val Cys Arg Ala Ala Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp Val
660 665 670
Gln Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala
675 680 685
Leu Ser Phe Thr Asn Leu Leu His Glu Ile Phe Asp Ile Glu Val Pro
690 695 700
Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Gln Ala Leu Ala Asp
705 710 715 720
Tyr Val Glu Ala Ala Arg Lys Pro Gly Ser Ser Arg Pro Thr Phe Ala
725 730 735
Ser Val His Gly Ala Ser Asn Gly Gln Val Thr Glu Val His Ala Gly
740 745 750
Asp Leu Ser Leu Asp Lys Phe Ile Asp Ala Ala Thr Leu Ala Glu Ala
755 760 765
Pro Arg Leu Pro Ala Ala Asn Thr Gln Val Arg Thr Val Leu Leu Thr
770 775 780
Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu
785 790 795 800
Arg Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu Val Arg Ala Lys
805 810 815
Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly
820 825 830
Asp Pro Glu Leu Leu Ala His Tyr Arg Ala Leu Ala Gly Asp His Leu
835 840 845
Glu Val Leu Ala Gly Asp Lys Gly Glu Ala Asp Leu Gly Leu Asp Arg
850 855 860
Gln Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Leu Ile Val Asp Pro
865 870 875 880
Ala Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu Phe Gly Pro
885 890 895
Asn Ala Leu Gly Thr Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys
900 905 910
Ile Lys Pro Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp Gln Ile
915 920 925
Pro Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg Val Ile Ser Ala
930 935 940
Thr Arg Ala Val Asp Asp Ser Tyr Ala Asn Gly Tyr Ser Asn Ser Lys
945 950 955 960
Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu
965 970 975
Pro Val Ala Val Phe Arg Cys Asp Met Ile Leu Ala Asp Thr Thr Trp
980 985 990
Ala Gly Gln Leu Asn Val Pro Asp Met Phe Thr Arg Met Ile Leu Ser
995 1000 1005
Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala Ala
1010 1015 1020
Asp Gly Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val Glu Phe
1025 1030 1035 1040
Ile Ala Glu Ala Ile Ser Thr Leu Gly Ala Gln Ser Gln Asp Gly Phe
1045 1050 1055
His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp
1060 1065 1070
Glu Phe Val Asp Trp Leu Asn Glu Ser Gly Cys Pro Ile Gln Arg Ile
1075 1080 1085
Ala Asp Tyr Gly Asp Trp Leu Gln Arg Phe Glu Thr Ala Leu Arg Ala
1090 1095 1100
Leu Pro Asp Arg Gln Arg His Ser Ser Leu Leu Pro Leu Leu His Asn
1105 1110 1115 1120
Tyr Arg Gln Pro Glu Arg Pro Val Arg Gly Ser Ile Ala Pro Thr Asp
1125 1130 1135
Arg Phe Arg Ala Ala Val Gln Glu Ala Lys Ile Gly Pro Asp Lys Asp
1140 1145 1150
Ile Pro His Val Gly Ala Pro Ile Ile Val Lys Tyr Val Ser Asp Leu
1155 1160 1165
Arg Leu Leu Gly Leu Leu
1170
<210> 3
<211> 1173
<212> PRT
<213>Mycobacterium smegmatis
<400> 3
Met Thr Ser Asp Val His Asp Ala Thr Asp Gly Val Thr Glu Thr Ala
1 5 10 15
Leu Asp Asp Glu Gln Ser Thr Arg Arg Ile Ala Glu Leu Tyr Ala Thr
20 25 30
Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp Ala
35 40 45
Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu Phe
50 55 60
Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu Leu
65 70 75 80
Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg Phe
85 90 95
Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala Val Ala
100 105 110
Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr Pro Gly Asp Ala
115 120 125
Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu Thr Leu Asp Leu
130 135 140
Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu Gln His Asn Ala
145 150 155 160
Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val Glu Pro Arg Ile
165 170 175
Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val Glu Ser Val Arg
180 185 190
Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp His His Pro Glu
195 200 205
Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg Glu Gln Leu Ala
210 215 220
Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile Ala Asp Glu Gly
225 230 235 240
Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp His Asp Gln Arg
245 250 255
Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly
260 265 270
Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser Phe
275 280 285
Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro Leu
290 295 300
Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala Val Gln Asn Gly
305 310 315 320
Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser Thr Leu Phe Glu
325 330 335
Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu Val Pro Arg Val
340 345 350
Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val Asp Arg Leu Val
355 360 365
Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala Glu
370 375 380
Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val Ser
385 390 395 400
Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr Leu
405 410 415
Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly Ala Val
420 425 430
Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile Asp Tyr Lys Leu
435 440 445
Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp Lys Pro Tyr Pro
450 455 460
Arg Gly Glu Leu Leu Val Arg Ser Gln Thr Leu Thr Pro Gly Tyr Tyr
465 470 475 480
Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr Tyr
485 490 495
His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val Tyr
500 505 510
Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val
515 520 525
Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala Ala Leu Val Arg
530 535 540
Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val
545 550 555 560
Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp Pro Ala Ala Leu
565 570 575
Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala Arg Asp Ala Glu
580 585 590
Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val Glu Thr Glu Pro
595 600 605
Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly Lys Leu Leu Arg
610 615 620
Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr Ala
625 630 635 640
Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg Ala
645 650 655
Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala Ala Thr
660 665 670
Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala His Phe Thr Asp
675 680 685
Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser Asn Leu Leu Ser
690 695 700
Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile Val Asn Pro Ala
705 710 715 720
Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala Gln Arg Thr Ala
725 730 735
Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala Thr
740 745 750
Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala Glu
755 760 765
Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro Arg
770 775 780
Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe Leu Thr
785 790 795 800
Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly Thr Leu Ile Thr
805 810 815
Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala Arg Leu Thr Gln
820 825 830
Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe Ala Glu Leu Ala
835 840 845
Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly Asp Pro Asn Leu
850 855 860
Gly Leu Thr Pro Glu Ile Trp His Arg Leu Ala Ala Glu Val Asp Leu
865 870 875 880
Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg Gln
885 890 895
Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val Ile Lys Leu Ala
900 905 910
Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ser Val
915 920 925
Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thr Val
930 935 940
Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn Gly Tyr Gly Asn
945 950 955 960
Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys
965 970 975
Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His Pro
980 985 990
Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu Leu
995 1000 1005
Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr Ile Gly
1010 1015 1020
Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly Leu Thr Val Asp Phe
1025 1030 1035 1040
Val Ala Glu Ala Val Thr Thr Leu Gly Ala Gln Gln Arg Glu Gly Tyr
1045 1050 1055
Val Ser Tyr Asp Val Met Asn Pro His Asp Asp Gly Ile Ser Leu Asp
1060 1065 1070
Val Phe Val Asp Trp Leu Ile Arg Ala Gly His Pro Ile Asp Arg Val
1075 1080 1085
Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe Glu Thr Ala Leu Thr Ala
1090 1095 1100
Leu Pro Glu Lys Arg Arg Ala Gln Thr Val Leu Pro Leu Leu His Ala
1105 1110 1115 1120
Phe Arg Ala Pro Gln Ala Pro Leu Arg Gly Ala Pro Glu Pro Thr Glu
1125 1130 1135
Val Phe His Ala Ala Val Arg Thr Ala Lys Val Gly Pro Gly Asp Ile
1140 1145 1150
Pro His Leu Asp Glu Ala Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg
1155 1160 1165
Glu Phe Gly Leu Ile
1170
<210> 4
<211> 1148
<212> PRT
<213> Segniliparus rugosus
<400> 4
Met Gly Asp Gly Glu Glu Arg Ala Lys Arg Phe Phe Gln Arg Ile Gly
1 5 10 15
Glu Leu Ser Ala Thr Asp Pro Gln Phe Ala Ala Ala Ala Pro Asp Pro
20 25 30
Ala Val Val Glu Ala Val Ser Asp Pro Ser Leu Ser Phe Thr Arg Tyr
35 40 45
Leu Asp Thr Leu Met Arg Gly Tyr Ala Glu Arg Pro Ala Leu Ala His
50 55 60
Arg Val Gly Ala Gly Tyr Glu Thr Ile Ser Tyr Gly Glu Leu Trp Ala
65 70 75 80
Arg Val Gly Ala Ile Ala Ala Ala Trp Gln Ala Asp Gly Leu Ala Pro
85 90 95
Gly Asp Phe Val Ala Thr Val Gly Phe Thr Ser Pro Asp Tyr Val Ala
100 105 110
Val Asp Leu Ala Ala Ala Arg Ser Gly Leu Val Ser Val Pro Leu Gln
115 120 125
Ala Gly Ala Ser Leu Ala Gln Leu Val Gly Ile Leu Glu Glu Thr Glu
130 135 140
Pro Lys Val Leu Ala Ala Ser Ala Ser Ser Leu Glu Gly Ala Val Ala
145 150 155 160
Cys Ala Leu Ala Ala Pro Ser Val Gln Arg Leu Val Val Phe Asp Leu
165 170 175
Arg Gly Pro Asp Ala Ser Glu Ser Ala Ala Asp Glu Arg Arg Gly Ala
180 185 190
Leu Ala Asp Ala Glu Glu Gln Leu Ala Arg Ala Gly Arg Ala Val Val
195 200 205
Val Glu Thr Leu Ala Asp Leu Ala Ala Arg Gly Glu Ala Leu Pro Glu
210 215 220
Ala Pro Leu Phe Glu Pro Ala Glu Gly Glu Asp Pro Leu Ala Leu Leu
225 230 235 240
Ile Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly Ala Met Tyr Ser
245 250 255
Gln Arg Leu Val Ser Gln Leu Trp Gly Arg Thr Pro Val Val Pro Gly
260 265 270
Met Pro Asn Ile Ser Leu His Tyr Met Pro Leu Ser His Ser Tyr Gly
275 280 285
Arg Ala Val Leu Ala Gly Ala Leu Ser Ala Gly Gly Thr Ala His Phe
290 295 300
Thr Ala Asn Ser Asp Leu Ser Thr Leu Phe Glu Asp Ile Ala Leu Ala
305 310 315 320
Arg Pro Thr Phe Leu Ala Leu Val Pro Arg Val Cys Glu Met Leu Phe
325 330 335
Gln Glu Ser Gln Arg Gly Gln Asp Val Ala Glu Leu Arg Glu Arg Val
340 345 350
Leu Gly Gly Arg Leu Leu Val Ala Val Cys Gly Ser Ala Pro Leu Ser
355 360 365
Pro Glu Met Arg Ala Phe Met Glu Glu Val Leu Gly Phe Pro Leu Leu
370 375 380
Asp Gly Tyr Gly Ser Thr Glu Ala Leu Gly Val Met Arg Asn Gly Ile
385 390 395 400
Ile Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Glu
405 410 415
Leu Gly Tyr Arg Thr Thr Asp Lys Pro Tyr Pro Arg Gly Glu Leu Cys
420 425 430
Ile Arg Ser Thr Ser Leu Ile Ser Gly Tyr Tyr Lys Arg Pro Glu Ile
435 440 445
Thr Ala Glu Val Phe Asp Ala Gln Gly Tyr Tyr Lys Thr Gly Asp Val
450 455 460
Met Ala Glu Ile Ala Pro Asp His Leu Val Tyr Val Asp Arg Ser Lys
465 470 475 480
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Lys Leu
485 490 495
Glu Ala Ala Tyr Gly Thr Ser Pro Tyr Val Lys Gln Ile Phe Val Tyr
500 505 510
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Asn Ala
515 520 525
Glu Val Leu Gly Ala Arg Asp Gln Glu Glu Ala Lys Pro Leu Ile Ala
530 535 540
Ala Ser Leu Gln Lys Ile Ala Lys Glu Ala Gly Leu Gln Ser Tyr Glu
545 550 555 560
Val Pro Arg Asp Phe Leu Ile Glu Thr Glu Pro Phe Thr Thr Gln Asn
565 570 575
Gly Leu Leu Ser Glu Val Gly Lys Leu Leu Arg Pro Lys Leu Lys Ala
580 585 590
Arg Tyr Gly Glu Ala Leu Glu Ala Arg Tyr Asp Glu Ile Ala His Gly
595 600 605
Gln Ala Asp Glu Leu Arg Ala Leu Arg Asp Gly Ala Gly Gln Arg Pro
610 615 620
Val Val Glu Thr Val Val Arg Ala Ala Val Ala Ile Ser Gly Ser Glu
625 630 635 640
Gly Ala Glu Val Gly Pro Glu Ala Asn Phe Ala Asp Leu Gly Gly Asp
645 650 655
Ser Leu Ser Ala Leu Ser Leu Ala Asn Leu Leu His Asp Val Phe Glu
660 665 670
Val Glu Val Pro Val Arg Ile Ile Ile Gly Pro Thr Ala Ser Leu Ala
675 680 685
Gly Ile Ala Lys His Ile Glu Ala Glu Arg Ala Gly Ala Ser Ala Pro
690 695 700
Thr Ala Ala Ser Val His Gly Ala Gly Ala Thr Arg Ile Arg Ala Ser
705 710 715 720
Glu Leu Thr Leu Glu Lys Phe Leu Pro Glu Asp Leu Leu Ala Ala Ala
725 730 735
Lys Gly Leu Pro Ala Ala Asp Gln Val Arg Thr Val Leu Leu Thr Gly
740 745 750
Ala Asn Gly Trp Leu Gly Arg Phe Leu Ala Leu Glu Gln Leu Glu Arg
755 760 765
Leu Ala Arg Ser Gly Gln Asp Gly Gly Lys Leu Ile Cys Leu Val Arg
770 775 780
Gly Lys Asp Ala Ala Ala Ala Arg Arg Arg Ile Glu Glu Thr Leu Gly
785 790 795 800
Thr Asp Pro Ala Leu Ala Ala Arg Phe Ala Glu Leu Ala Glu Gly Arg
805 810 815
Leu Glu Val Val Pro Gly Asp Val Gly Glu Pro Lys Phe Gly Leu Asp
820 825 830
Asp Ala Ala Trp Asp Arg Leu Ala Glu Glu Val Asp Val Ile Val His
835 840 845
Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr His Gln Leu Phe Gly
850 855 860
Pro Asn Val Val Gly Thr Ala Glu Ile Ile Arg Leu Ala Ile Thr Ala
865 870 875 880
Lys Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly
885 890 895
Val Glu Pro Ser Ser Phe Glu Glu Asp Gly Asp Ile Arg Ala Val Val
900 905 910
Pro Glu Arg Pro Leu Gly Asp Gly Tyr Ala Asn Gly Tyr Gly Asn Ser
915 920 925
Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Glu Leu Val Gly
930 935 940
Leu Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His Thr Arg
945 950 955 960
Tyr Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu Val Leu
965 970 975
Ser Leu Leu Ala Thr Gly Ile Ala Pro Lys Ser Phe Tyr Gln Gln Gly
980 985 990
Ala Ala Gly Glu Arg Gln Arg Ala His Tyr Asp Gly Ile Pro Val Asp
995 1000 1005
Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Ala Glu Pro Ser Trp Phe
1010 1015 1020
Asp Gly Gly Ala Gly Phe Arg Ser Phe Asp Val Phe Asn Pro His His
1025 1030 1035 1040
Asp Gly Val Gly Leu Asp Glu Phe Val Asp Trp Leu Ile Glu Ala Gly
1045 1050 1055
His Pro Ile Ser Arg Ile Asp Asp His Lys Glu Trp Phe Ala Arg Phe
1060 1065 1070
Glu Thr Ala Val Arg Gly Leu Pro Glu Ala Gln Arg Gln His Ser Leu
1075 1080 1085
Leu Pro Leu Leu Arg Ala Tyr Ser Phe Pro His Pro Pro Val Asp Gly
1090 1095 1100
Ser Val Tyr Pro Thr Gly Lys Phe Gln Gly Ala Val Lys Ala Ala Gln
1105 1110 1115 1120
Val Gly Ser Asp His Asp Val Pro His Leu Gly Lys Ala Leu Ile Val
1125 1130 1135
Lys Tyr Ala Asp Asp Leu Lys Ala Leu Gly Leu Leu
1140 1145
<210> 5
<211> 1168
<212> PRT
<213>Mycobacterium smegmatis
<400> 5
Met Thr Ile Glu Thr Arg Glu Asp Arg Phe Asn Arg Arg Ile Asp His
1 5 10 15
Leu Phe Glu Thr Asp Pro Gln Phe Ala Ala Ala Arg Pro Asp Glu Ala
20 25 30
Ile Ser Ala Ala Ala Ala Asp Pro Glu Leu Arg Leu Pro Ala Ala Val
35 40 45
Lys Gln Ile Leu Ala Gly Tyr Ala Asp Arg Pro Ala Leu Gly Lys Arg
50 55 60
Ala Val Glu Phe Val Thr Asp Glu Glu Gly Arg Thr Thr Ala Lys Leu
65 70 75 80
Leu Pro Arg Phe Asp Thr Ile Thr Tyr Arg Gln Leu Ala Gly Arg Ile
85 90 95
Gln Ala Val Thr Asn Ala Trp His Asn His Pro Val Asn Ala Gly Asp
100 105 110
Arg Val Ala Ile Leu Gly Phe Thr Ser Val Asp Tyr Thr Thr Ile Asp
115 120 125
Ile Ala Leu Leu Glu Leu Gly Ala Val Ser Val Pro Leu Gln Thr Ser
130 135 140
Ala Pro Val Ala Gln Leu Gln Pro Ile Val Ala Glu Thr Glu Pro Lys
145 150 155 160
Val Ile Ala Ser Ser Val Asp Phe Leu Ala Asp Ala Val Ala Leu Val
165 170 175
Glu Ser Gly Pro Ala Pro Ser Arg Leu Val Val Phe Asp Tyr Ser His
180 185 190
Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala Ala Lys Gly Lys Leu
195 200 205
Ala Gly Thr Gly Val Val Val Glu Thr Ile Thr Asp Ala Leu Asp Arg
210 215 220
Gly Arg Ser Leu Ala Asp Ala Pro Leu Tyr Val Pro Asp Glu Ala Asp
225 230 235 240
Pro Leu Thr Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
245 250 255
Gly Ala Met Tyr Pro Glu Ser Lys Thr Ala Thr Met Trp Gln Ala Gly
260 265 270
Ser Lys Ala Arg Trp Asp Glu Thr Leu Gly Val Met Pro Ser Ile Thr
275 280 285
Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gly Ile Leu Cys
290 295 300
Ser Thr Leu Ala Ser Gly Gly Thr Ala Tyr Phe Ala Ala Arg Ser Asp
305 310 315 320
Leu Ser Thr Phe Leu Glu Asp Leu Ala Leu Val Arg Pro Thr Gln Leu
325 330 335
Asn Phe Val Pro Arg Ile Trp Asp Met Leu Phe Gln Glu Tyr Gln Ser
340 345 350
Arg Leu Asp Asn Arg Arg Ala Glu Gly Ser Glu Asp Arg Ala Glu Ala
355 360 365
Ala Val Leu Glu Glu Val Arg Thr Gln Leu Leu Gly Gly Arg Phe Val
370 375 380
Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Ala Glu Met Lys Ser Trp
385 390 395 400
Val Glu Asp Leu Leu Asp Met His Leu Leu Glu Gly Tyr Gly Ser Thr
405 410 415
Glu Ala Gly Ala Val Phe Ile Asp Gly Gln Ile Gln Arg Pro Pro Val
420 425 430
Ile Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe Ala Thr
435 440 445
Asp Arg Pro Tyr Pro Arg Gly Glu Leu Leu Val Lys Ser Glu Gln Met
450 455 460
Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Ile Thr Ala Glu Met Phe Asp
465 470 475 480
Glu Asp Gly Tyr Tyr Arg Thr Gly Asp Ile Val Ala Glu Leu Gly Pro
485 490 495
Asp His Leu Glu Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys Leu Ser
500 505 510
Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe Gly Asp
515 520 525
Ser Pro Leu Val Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala Arg Ser
530 535 540
Tyr Leu Leu Ala Val Val Val Pro Thr Glu Glu Ala Leu Ser Arg Trp
545 550 555 560
Asp Gly Asp Glu Leu Lys Ser Arg Ile Ser Asp Ser Leu Gln Asp Ala
565 570 575
Ala Arg Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp Phe Leu
580 585 590
Val Glu Thr Thr Pro Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile
595 600 605
Arg Lys Leu Ala Arg Pro Lys Leu Lys Ala His Tyr Gly Glu Arg Leu
610 615 620
Glu Gln Leu Tyr Thr Asp Leu Ala Glu Gly Gln Ala Asn Glu Leu Arg
625 630 635 640
Glu Leu Arg Arg Asn Gly Ala Asp Arg Pro Val Val Glu Thr Val Ser
645 650 655
Arg Ala Ala Val Ala Leu Leu Gly Ala Ser Val Thr Asp Leu Arg Ser
660 665 670
Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser
675 680 685
Phe Ser Asn Leu Leu His Glu Ile Phe Asp Val Asp Val Pro Val Gly
690 695 700
Val Ile Val Ser Pro Ala Thr Asp Leu Ala Gly Val Ala Ala Tyr Ile
705 710 715 720
Glu Gly Glu Leu Arg Gly Ser Lys Arg Pro Thr Tyr Ala Ser Val His
725 730 735
Gly Arg Asp Ala Thr Glu Val Arg Ala Arg Asp Leu Ala Leu Gly Lys
740 745 750
Phe Ile Asp Ala Lys Thr Leu Ser Ala Ala Pro Gly Leu Pro Arg Ser
755 760 765
Gly Thr Glu Ile Arg Thr Val Leu Leu Thr Gly Ala Thr Gly Phe Leu
770 775 780
Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met Asp Leu Val Asp
785 790 795 800
Gly Lys Val Ile Cys Leu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg
805 810 815
Ala Arg Leu Asp Ala Thr Phe Asp Thr Gly Asp Ala Thr Leu Leu Glu
820 825 830
His Tyr Arg Ala Leu Ala Ala Asp His Leu Glu Val Ile Ala Gly Asp
835 840 845
Lys Gly Glu Ala Asp Leu Gly Leu Asp His Asp Thr Trp Gln Arg Leu
850 855 860
Ala Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala Leu Val Asn His
865 870 875 880
Val Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Leu Gly Thr Ala
885 890 895
Glu Leu Ile Arg Ile Ala Leu Thr Thr Thr Ile Lys Pro Tyr Val Tyr
900 905 910
Val Ser Thr Ile Gly Val Gly Gln Gly Ile Ser Pro Glu Ala Phe Val
915 920 925
Glu Asp Ala Asp Ile Arg Glu Ile Ser Ala Thr Arg Arg Val Asp Asp
930 935 940
Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu
945 950 955 960
Leu Arg Glu Ala His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg
965 970 975
Cys Asp Met Ile Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu
980 985 990
Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr Gly Ile
995 1000 1005
Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn Arg Gln Arg
1010 1015 1020
Ala His Tyr Asp Gly Leu Pro Val Glu Phe Ile Ala Glu Ala Ile Ser
1025 1030 1035 1040
Thr Ile Gly Ser Gln Val Thr Asp Gly Phe Glu Thr Phe His Val Met
1045 1050 1055
Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp Glu Tyr Val Asp Trp Leu
1060 1065 1070
Ile Glu Ala Gly Tyr Pro Val His Arg Val Asp Asp Tyr Ala Thr Trp
1075 1080 1085
Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala Leu Pro Glu Arg Gln Arg
1090 1095 1100
Gln Ala Ser Leu Leu Pro Leu Leu His Asn Tyr Gln Gln Pro Ser Pro
1105 1110 1115 1120
Pro Val Cys Gly Ala Met Ala Pro Thr Asp Arg Phe Arg Ala Ala Val
1125 1130 1135
Gln Asp Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Thr Ala
1140 1145 1150
Asp Val Ile Val Lys Tyr Ile Ser Asn Leu Gln Met Leu Gly Leu Leu
1155 1160 1165
<210> 6
<211> 1185
<212> PRT
<213>Marseille mycobacteria
<400> 6
Met Thr Asn Glu Thr Asn Pro Gln Gln Glu Gln Leu Ser Arg Arg Ile
1 5 10 15
Glu Ser Leu Arg Glu Ser Asp Pro Gln Phe Arg Ala Ala Gln Pro Asp
20 25 30
Pro Ala Val Ala Glu Gln Val Leu Arg Pro Gly Leu His Leu Ser Glu
35 40 45
Ala Ile Ala Ala Leu Met Thr Gly Tyr Ala Glu Arg Pro Ala Leu Gly
50 55 60
Glu Arg Ala Arg Glu Leu Val Ile Asp Gln Asp Gly Arg Thr Thr Leu
65 70 75 80
Arg Leu Leu Pro Arg Phe Asp Thr Thr Thr Tyr Gly Glu Leu Trp Ser
85 90 95
Arg Thr Thr Ser Val Ala Ala Ala Trp His His Asp Ala Thr His Pro
100 105 110
Val Lys Ala Gly Asp Leu Val Ala Thr Leu Gly Phe Thr Ser Ile Asp
115 120 125
Tyr Thr Val Leu Asp Leu Ala Ile Met Ile Leu Gly Gly Val Ala Val
130 135 140
Pro Leu Gln Thr Ser Ala Pro Ala Ser Gln Trp Thr Thr Ile Leu Ala
145 150 155 160
Glu Ala Glu Pro Asn Thr Leu Ala Val Ser Ile Glu Leu Ile Gly Ala
165 170 175
Ala Met Glu Ser Val Arg Ala Thr Pro Ser Ile Lys Gln Val Val Val
180 185 190
Phe Asp Tyr Thr Pro Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala
195 200 205
Ala Ser Thr Gln Leu Ala Gly Thr Gly Ile Ala Leu Glu Thr Leu Asp
210 215 220
Ala Val Ile Ala Arg Gly Ala Ala Leu Pro Ala Ala Pro Leu Tyr Ala
225 230 235 240
Pro Ser Ala Gly Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser Gly
245 250 255
Ser Thr Gly Ala Pro Lys Gly Ala Met His Ser Glu Asn Ile Val Arg
260 265 270
Arg Trp Trp Ile Arg Glu Asp Val Met Ala Gly Thr Glu Asn Leu Pro
275 280 285
Met Ile Gly Leu Asn Phe Met Pro Met Ser His Ile Met Gly Arg Gly
290 295 300
Thr Leu Thr Ser Thr Leu Ser Thr Gly Gly Thr Gly Tyr Phe Ala Ala
305 310 315 320
Ser Ser Asp Met Ser Thr Leu Phe Glu Asp Met Glu Leu Ile Arg Pro
325 330 335
Thr Ala Leu Ala Leu Val Pro Arg Val Cys Asp Met Val Phe Gln Arg
340 345 350
Phe Gln Thr Glu Val Asp Arg Arg Leu Ala Ser Gly Asp Thr Ala Ser
355 360 365
Ala Glu Ala Val Ala Ala Glu Val Lys Ala Asp Ile Arg Asp Asn Leu
370 375 380
Phe Gly Gly Arg Val Ser Ala Val Met Val Gly Ser Ala Pro Leu Ser
385 390 395 400
Glu Glu Leu Gly Glu Phe Ile Glu Ser Cys Phe Glu Leu Asn Leu Thr
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Met Val Phe Arg Asp Gly Ile
420 425 430
Val Gln Arg Pro Pro Val Ile Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Ser Thr Asp Lys Pro His Pro Arg Gly Glu Leu Leu
450 455 460
Leu Lys Thr Asp Gly Met Phe Leu Gly Tyr Tyr Lys Arg Pro Glu Val
465 470 475 480
Thr Ala Ser Val Phe Asp Ala Asp Gly Phe Tyr Met Thr Gly Asp Ile
485 490 495
Val Ala Glu Leu Ala His Asp Asn Ile Glu Ile Ile Asp Arg Arg Asn
500 505 510
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Ala Val Ala Thr Leu
515 520 525
Glu Ala Glu Tyr Ala Asn Ser Pro Val Val His Gln Ile Tyr Val Tyr
530 535 540
Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Ala Val Ala Ala Ala Lys Gly Asp Ala Ala Ala Leu Lys Thr Thr
565 570 575
Ile Ala Asp Ser Leu Gln Asp Ile Ala Lys Glu Ile Gln Leu Gln Ser
580 585 590
Tyr Glu Val Pro Arg Asp Phe Ile Ile Glu Pro Gln Pro Phe Thr Gln
595 600 605
Gly Asn Gly Leu Leu Thr Gly Ile Ala Lys Leu Ala Arg Pro Asn Leu
610 615 620
Lys Ala His Tyr Gly Pro Arg Leu Glu Gln Met Tyr Ala Glu Ile Ala
625 630 635 640
Glu Gln Gln Ala Ala Glu Leu Arg Ala Leu His Gly Val Asp Pro Asp
645 650 655
Lys Pro Ala Leu Glu Thr Val Leu Lys Ala Ala Gln Ala Leu Leu Gly
660 665 670
Val Ser Ser Ala Glu Leu Ala Ala Asp Ala His Phe Thr Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Phe Ser Asp Leu Leu Arg Asp Ile
690 695 700
Phe Ala Val Glu Val Pro Val Gly Val Ile Val Ser Ala Ala Asn Asp
705 710 715 720
Leu Gly Gly Val Ala Lys Phe Val Asp Glu Gln Arg His Ser Gly Gly
725 730 735
Thr Arg Pro Thr Ala Glu Thr Val His Gly Ala Gly His Thr Glu Ile
740 745 750
Arg Ala Ala Asp Leu Thr Leu Asp Lys Phe Ile Asp Glu Ala Thr Leu
755 760 765
His Ala Ala Pro Ser Leu Pro Lys Ala Ala Gly Ile Pro His Thr Val
770 775 780
Leu Leu Thr Gly Ser Asn Gly Tyr Leu Gly His Tyr Leu Ala Leu Glu
785 790 795 800
Trp Leu Glu Arg Leu Asp Lys Thr Asp Gly Lys Leu Ile Val Ile Val
805 810 815
Arg Gly Lys Asn Ala Glu Ala Ala Tyr Gly Arg Leu Glu Glu Ala Phe
820 825 830
Asp Thr Gly Asp Thr Glu Leu Leu Ala His Phe Arg Ser Leu Ala Asp
835 840 845
Lys His Leu Glu Val Leu Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly
850 855 860
Leu Asp Ala Asp Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Val Ile
865 870 875 880
Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Asn Gln Leu
885 890 895
Phe Gly Pro Asn Val Val Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Thr Thr Lys Ile Lys Pro Val Thr Tyr Leu Ser Thr Val Ala Val Ala
915 920 925
Ala Tyr Val Asp Pro Thr Thr Phe Asp Glu Glu Ser Asp Ile Arg Leu
930 935 940
Ile Ser Ala Val Arg Pro Ile Asp Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ala Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Cys Gly Leu Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Arg Tyr Thr Gly Gln Leu Asn Val Pro Asp Gln Phe Thr Arg Leu
995 1000 1005
Ile Leu Ser Leu Ile Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Gln
1010 1015 1020
Ala Gln Thr Thr Gly Glu Arg Pro Leu Ala His Tyr Asp Gly Leu Pro
1025 1030 1035 1040
Gly Asp Phe Thr Ala Glu Ala Ile Thr Thr Leu Gly Thr Gln Val Pro
1045 1050 1055
Glu Gly Ser Glu Gly Phe Val Thr Tyr Asp Cys Val Asn Pro His Ala
1060 1065 1070
Asp Gly Ile Ser Leu Asp Asn Phe Val Asp Trp Leu Ile Glu Ala Gly
1075 1080 1085
Tyr Pro Ile Ala Arg Ile Asp Asn Tyr Thr Glu Trp Phe Thr Arg Phe
1090 1095 1100
Asp Thr Ala Ile Arg Gly Leu Ser Glu Lys Gln Lys Gln His Ser Leu
1105 1110 1115 1120
Leu Pro Leu Leu His Ala Phe Glu Gln Pro Ser Ala Ala Glu Asn His
1125 1130 1135
Gly Val Val Pro Ala Lys Arg Phe Gln His Ala Val Gln Ala Ala Gly
1140 1145 1150
Ile Gly Pro Val Gly Gln Asp Gly Thr Thr Asp Ile Pro His Leu Ser
1155 1160 1165
Arg Arg Leu Ile Val Lys Tyr Ala Lys Asp Leu Glu Gln Leu Gly Leu
1170 1175 1180
Leu
1185
<210> 7
<211> 1186
<212> PRT
<213> Segniliparus rotundus
<400> 7
Met Thr Gln Ser His Thr Gln Gly Pro Gln Ala Ser Ala Ala His Ser
1 5 10 15
Arg Leu Ala Arg Arg Ala Ala Glu Leu Leu Ala Thr Asp Pro Gln Ala
20 25 30
Ala Ala Thr Leu Pro Asp Pro Glu Val Val Arg Gln Ala Thr Arg Pro
35 40 45
Gly Leu Arg Leu Ala Glu Arg Val Asp Ala Ile Leu Ser Gly Tyr Ala
50 55 60
Asp Arg Pro Ala Leu Gly Gln Arg Ser Phe Gln Thr Val Lys Asp Pro
65 70 75 80
Ile Thr Gly Arg Ser Ser Val Glu Leu Leu Pro Thr Phe Asp Thr Ile
85 90 95
Thr Tyr Arg Glu Leu Arg Glu Arg Ala Thr Ala Ile Ala Ser Asp Leu
100 105 110
Ala His His Pro Gln Ala Pro Ala Lys Pro Gly Asp Phe Leu Ala Ser
115 120 125
Ile Gly Phe Ile Ser Val Asp Tyr Val Ala Ile Asp Ile Ala Gly Val
130 135 140
Phe Ala Gly Leu Thr Ala Val Pro Leu Gln Thr Gly Ala Thr Leu Ala
145 150 155 160
Thr Leu Thr Ala Ile Thr Ala Glu Thr Ala Pro Thr Leu Phe Ala Ala
165 170 175
Ser Ile Glu His Leu Pro Thr Ala Val Asp Ala Val Leu Ala Thr Pro
180 185 190
Ser Val Arg Arg Leu Leu Val Phe Asp Tyr Arg Ala Gly Ser Asp Glu
195 200 205
Asp Arg Glu Ala Val Glu Ala Ala Lys Arg Lys Ile Ala Asp Ala Gly
210 215 220
Ser Ser Val Leu Val Asp Val Leu Asp Glu Val Ile Ala Arg Gly Lys
225 230 235 240
Ser Ala Pro Lys Ala Pro Leu Pro Pro Ala Thr Asp Ala Gly Asp Asp
245 250 255
Ser Leu Ser Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys
260 265 270
Gly Ala Met Tyr Pro Glu Arg Asn Val Ala His Phe Trp Gly Gly Val
275 280 285
Trp Ala Ala Ala Phe Asp Glu Asp Ala Ala Pro Pro Val Pro Ala Ile
290 295 300
Asn Ile Thr Phe Leu Pro Leu Ser His Val Ala Ser Arg Leu Ser Leu
305 310 315 320
Met Pro Thr Leu Ala Arg Gly Gly Leu Met His Phe Val Ala Lys Ser
325 330 335
Asp Leu Ser Thr Leu Phe Glu Asp Leu Lys Leu Ala Arg Pro Thr Asn
340 345 350
Leu Phe Leu Val Pro Arg Val Val Glu Met Leu Tyr Gln His Tyr Gln
355 360 365
Ser Glu Leu Asp Arg Arg Gly Val Gln Asp Gly Thr Arg Glu Ala Glu
370 375 380
Ala Val Lys Asp Asp Leu Arg Thr Gly Leu Leu Gly Gly Arg Ile Leu
385 390 395 400
Thr Ala Gly Phe Gly Ser Ala Pro Leu Ser Ala Glu Leu Ala Gly Phe
405 410 415
Ile Glu Ser Leu Leu Gln Ile His Leu Val Asp Gly Tyr Gly Ser Thr
420 425 430
Glu Ala Gly Pro Val Trp Arg Asp Gly Tyr Leu Val Lys Pro Pro Val
435 440 445
Thr Asp Tyr Lys Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr
450 455 460
Asp Ser Pro His Pro Arg Gly Glu Leu Ala Ile Lys Thr Gln Thr Ile
465 470 475 480
Leu Pro Gly Tyr Tyr Lys Arg Pro Glu Thr Thr Ala Glu Val Phe Asp
485 490 495
Glu Asp Gly Phe Tyr Leu Thr Gly Asp Val Val Ala Gln Ile Gly Pro
500 505 510
Glu Gln Phe Ala Tyr Val Asp Arg Arg Lys Asn Val Leu Lys Leu Ser
515 520 525
Gln Gly Glu Phe Val Thr Leu Ala Lys Leu Glu Ala Ala Tyr Ser Ser
530 535 540
Ser Pro Leu Val Arg Gln Leu Phe Val Tyr Gly Ser Ser Glu Arg Ser
545 550 555 560
Tyr Leu Leu Ala Val Ile Val Pro Thr Pro Asp Ala Leu Lys Lys Phe
565 570 575
Gly Val Gly Glu Ala Ala Lys Ala Ala Leu Gly Glu Ser Leu Gln Lys
580 585 590
Ile Ala Arg Asp Glu Gly Leu Gln Ser Tyr Glu Val Pro Arg Asp Phe
595 600 605
Ile Ile Glu Thr Asp Pro Phe Thr Val Glu Asn Gly Leu Leu Ser Asp
610 615 620
Ala Arg Lys Ser Leu Arg Pro Lys Leu Lys Glu His Tyr Gly Glu Arg
625 630 635 640
Leu Glu Ala Met Tyr Lys Glu Leu Ala Asp Gly Gln Ala Asn Glu Leu
645 650 655
Arg Asp Ile Arg Arg Gly Val Gln Gln Arg Pro Thr Leu Glu Thr Val
660 665 670
Arg Arg Ala Ala Ala Ala Met Leu Gly Ala Ser Ala Ala Glu Ile Lys
675 680 685
Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu
690 695 700
Thr Phe Ser Asn Phe Leu His Asp Leu Phe Glu Val Asp Val Pro Val
705 710 715 720
Gly Val Ile Val Ser Ala Ala Asn Thr Leu Gly Ser Val Ala Glu His
725 730 735
Ile Asp Ala Gln Leu Ala Gly Gly Arg Ala Arg Pro Thr Phe Ala Thr
740 745 750
Val His Gly Lys Gly Ser Thr Thr Ile Lys Ala Ser Asp Leu Thr Leu
755 760 765
Asp Lys Phe Ile Asp Glu Gln Thr Leu Glu Ala Ala Lys His Leu Pro
770 775 780
Lys Pro Ala Asp Pro Pro Arg Thr Val Leu Leu Thr Gly Ala Asn Gly
785 790 795 800
Trp Leu Gly Arg Phe Leu Ala Leu Glu Trp Leu Glu Arg Leu Ala Pro
805 810 815
Ala Gly Gly Lys Leu Ile Thr Ile Val Arg Gly Lys Asp Ala Ala Gln
820 825 830
Ala Lys Ala Arg Leu Asp Ala Ala Tyr Glu Ser Gly Asp Pro Lys Leu
835 840 845
Ala Gly His Tyr Gln Asp Leu Ala Ala Thr Thr Leu Glu Val Leu Ala
850 855 860
Gly Asp Phe Ser Glu Pro Arg Leu Gly Leu Asp Glu Ala Thr Trp Asn
865 870 875 880
Arg Leu Ala Asp Glu Val Asp Phe Ile Ser His Pro Gly Ala Leu Val
885 890 895
Asn His Val Leu Pro Tyr Asn Gln Leu Phe Gly Pro Asn Val Ala Gly
900 905 910
Val Ala Glu Ile Ile Lys Leu Ala Ile Thr Thr Arg Ile Lys Pro Val
915 920 925
Thr Tyr Leu Ser Thr Val Ala Val Ala Ala Gly Val Glu Pro Ser Ala
930 935 940
Leu Asp Glu Asp Gly Asp Ile Arg Thr Val Ser Ala Glu Arg Ser Val
945 950 955 960
Asp Glu Gly Tyr Ala Asn Gly Tyr Gly Asn Ser Lys Trp Gly Gly Glu
965 970 975
Val Leu Leu Arg Glu Ala His Asp Arg Thr Gly Leu Pro Val Arg Val
980 985 990
Phe Arg Ser Asp Met Ile Leu Ala His Gln Lys Tyr Thr Gly Gln Val
995 1000 1005
Asn Ala Thr Asp Gln Phe Thr Arg Leu Val Gln Ser Leu Leu Ala Thr
1010 1015 1020
Gly Leu Ala Pro Lys Ser Phe Tyr Glu Leu Asp Ala Gln Gly Asn Arg
1025 1030 1035 1040
Gln Arg Ala His Tyr Asp Gly Ile Pro Val Asp Phe Thr Ala Glu Ser
1045 1050 1055
Ile Thr Thr Leu Gly Gly Asp Gly Leu Glu Gly Tyr Arg Ser Tyr Asn
1060 1065 1070
Val Phe Asn Pro His Arg Asp Gly Val Gly Leu Asp Glu Phe Val Asp
1075 1080 1085
Trp Leu Ile Glu Ala Gly His Pro Ile Thr Arg Ile Asp Asp Tyr Asp
1090 1095 1100
Gln Trp Leu Ser Arg Phe Glu Thr Ser Leu Arg Gly Leu Pro Glu Ser
1105 1110 1115 1120
Lys Arg Gln Ala Ser Val Leu Pro Leu Leu His Ala Phe Ala Arg Pro
1125 1130 1135
Gly Pro Ala Val Asp Gly Ser Pro Phe Arg Asn Thr Val Phe Arg Thr
1140 1145 1150
Asp Val Gln Lys Ala Lys Ile Gly Ala Glu His Asp Ile Pro His Leu
1155 1160 1165
Gly Lys Ala Leu Val Leu Lys Tyr Ala Asp Asp Ile Lys Gln Leu Gly
1170 1175 1180
Leu Leu
1185
<210> 8
<211> 459
<212> PRT
<213>Blue or green chromabacterium biolaceum
<400> 8
Met Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Phe Phe Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Arg
405 410 415
Ala Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
<210> 9
<211> 468
<212> PRT
<213>Pseudomonas aeruginosa
<400> 9
Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu
1 5 10 15
Val Arg Ala Asp Gln Ala His Tyr Met His Gly Tyr His Val Phe Asp
20 25 30
Asp His Arg Val Asn Gly Ser Leu Asn Ile Ala Ala Gly Asp Gly Ala
35 40 45
Tyr Ile Tyr Asp Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly
50 55 60
Met Trp Cys Thr Asn Ile Gly Leu Gly Arg Glu Glu Met Ala Arg Thr
65 70 75 80
Val Ala Glu Gln Thr Arg Leu Leu Ala Tyr Ser Asn Pro Phe Cys Asp
85 90 95
Met Ala Asn Pro Arg Ala Ile Glu Leu Cys Arg Lys Leu Ala Glu Leu
100 105 110
Ala Pro Gly Asp Leu Asp His Val Phe Leu Thr Thr Gly Gly Ser Thr
115 120 125
Ala Val Asp Thr Ala Ile Arg Leu Met His Tyr Tyr Gln Asn Cys Arg
130 135 140
Gly Lys Arg Ala Lys Lys His Val Ile Thr Arg Ile Asn Ala Tyr His
145 150 155 160
Gly Ser Thr Phe Leu Gly Met Ser Leu Gly Gly Lys Ser Ala Asp Arg
165 170 175
Pro Ala Glu Phe Asp Phe Leu Asp Glu Arg Ile His His Leu Ala Cys
180 185 190
Pro Tyr Tyr Tyr Arg Ala Pro Glu Gly Leu Gly Glu Ala Glu Phe Leu
195 200 205
Asp Gly Leu Val Asp Glu Phe Glu Arg Lys Ile Leu Glu Leu Gly Ala
210 215 220
Asp Arg Val Gly Ala Phe Ile Ser Glu Pro Val Phe Gly Ser Gly Gly
225 230 235 240
Val Ile Val Pro Pro Ala Gly Tyr His Arg Arg Met Trp Glu Leu Cys
245 250 255
Gln Arg Tyr Asp Val Leu Tyr Ile Ser Asp Glu Val Val Thr Ser Phe
260 265 270
Gly Arg Leu Gly His Phe Phe Ala Ser Gln Ala Val Phe Gly Val Gln
275 280 285
Pro Asp Ile Ile Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr Gln Pro
290 295 300
Leu Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu Val Ile Ala Glu
305 310 315 320
Pro Asp Lys Gly Arg Cys Phe Ser His Gly Phe Thr Tyr Ser Gly His
325 330 335
Pro Val Ala Cys Ala Ala Ala Leu Lys Asn Ile Glu Ile Ile Glu Arg
340 345 350
Glu Gly Leu Leu Ala His Ala Asp Glu Val Gly Arg Tyr Phe Glu Glu
355 360 365
Arg Leu Gln Ser Leu Arg Asp Leu Pro Ile Val Gly Asp Val Arg Gly
370 375 380
Met Arg Phe Met Ala Cys Val Glu Phe Val Ala Asp Lys Ala Ser Lys
385 390 395 400
Ala Leu Phe Pro Glu Ser Leu Asn Ile Gly Glu Trp Val His Leu Arg
405 410 415
Ala Gln Lys Arg Gly Leu Leu Val Arg Pro Ile Val His Leu Asn Val
420 425 430
Met Ser Pro Pro Leu Ile Leu Thr Arg Glu Gln Val Asp Thr Val Val
435 440 445
Arg Val Leu Arg Glu Ser Ile Glu Glu Thr Val Glu Asp Leu Val Arg
450 455 460
Ala Gly His Arg
465
<210> 10
<211> 454
<212> PRT
<213>Pseudomonas syringae
<400> 10
Met Ser Ala Asn Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser
1 5 10 15
Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys
20 25 30
Gly Pro Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser
35 40 45
Glu Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala
50 55 60
Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gln Met
65 70 75 80
Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro Pro
85 90 95
Val Leu Glu Leu Ala Lys Ala Ile Ser Asp Ile Ala Pro Glu Gly Met
100 105 110
Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met
115 120 125
Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gln Pro Asn Lys
130 135 140
Lys Thr Ile Ile Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala
145 150 155 160
Gly Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu
165 170 175
Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu
180 185 190
Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala Glu Gln
195 200 205
Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe
210 215 220
Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Asp
225 230 235 240
Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser Arg Tyr Asp Ile Leu
245 250 255
Phe Ala Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Ser Glu Trp
260 265 270
Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr Ile Ala
275 280 285
Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg
290 295 300
Asp Glu Ile Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly
305 310 315 320
Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn
325 330 335
Ile Arg Ile Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg Ser Glu
340 345 350
Thr Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu Ser Asp His Pro
355 360 365
Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala Ile Glu Leu
370 375 380
Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly
385 390 395 400
Met Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala
405 410 415
Val Gly Asp Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala
420 425 430
Gln Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr
435 440 445
Leu Ala Val Leu Gln Gly
450
<210> 11
<211> 467
<212> PRT
<213>Rhodobacter
<400> 11
Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly Ala Val Gly Ala Ala
1 5 10 15
Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu Met Ala Lys Leu Gly
20 25 30
Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu Gly Ile Tyr Val His
35 40 45
Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro Ala Gly Met Trp Cys
50 55 60
Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val Asp Ala Met Ala His
65 70 75 80
Gln Ala Met Val Leu Pro Tyr Ala Ser Pro Trp Tyr Met Ala Thr Ser
85 90 95
Pro Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr Leu Thr Pro Gly Asp
100 105 110
Leu Asn Arg Ile Phe Phe Thr Thr Gly Gly Ser Thr Ala Val Asp Ser
115 120 125
Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val Leu Gly Arg Pro Gln
130 135 140
Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr His Gly Ser Thr Ala
145 150 155 160
Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn Trp Pro Asn Phe Asp
165 170 175
Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser Pro Asn Pro Arg His
180 185 190
Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp Asp Leu Val Gln Glu
195 200 205
Phe Glu Asp Arg Ile Glu Ser Leu Gly Pro Asp Thr Ile Ala Ala Phe
210 215 220
Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val Ile Ile Pro Pro Ala
225 230 235 240
Gly Tyr His Ala Arg Phe Lys Ala Ile Cys Glu Lys His Asp Ile Leu
245 250 255
Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly Arg Cys Gly Glu Trp
260 265 270
Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro Asp Ile Ile Thr Phe
275 280 285
Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu Gly Gly Leu Ala Ile
290 295 300
Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu Asn Ala Lys Gly Ser
305 310 315 320
Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gln Pro Val Ala Cys Ala
325 330 335
Ala Ala Leu Ala Asn Ile Glu Leu Met Glu Arg Glu Gly Ile Val Asp
340 345 350
Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala Ala Leu Ala Ser Leu
355 360 365
Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser Val Gly Leu Val Gly
370 375 380
Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala Asp Gly Thr Ala Glu
385 390 395 400
Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg Cys Phe Glu Leu Gly
405 410 415
Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val Ile Ser Pro Pro Leu
420 425 430
Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val Ala Ile Met Arg Gln
435 440 445
Ala Ile Thr Glu Val Ser Ala Ala His Gly Leu Thr Ala Lys Glu Pro
450 455 460
Ala Ala Val
465
<210> 12
<211> 459
<212> PRT
<213>Escherichia coli
<400> 12
Met Asn Arg Leu Pro Ser Ser Ala Ser Ala Leu Ala Cys Ser Ala His
1 5 10 15
Ala Leu Asn Leu Ile Glu Lys Arg Thr Leu Asp His Glu Glu Met Lys
20 25 30
Ala Leu Asn Arg Glu Val Ile Glu Tyr Phe Lys Glu His Val Asn Pro
35 40 45
Gly Phe Leu Glu Tyr Arg Lys Ser Val Thr Ala Gly Gly Asp Tyr Gly
50 55 60
Ala Val Glu Trp Gln Ala Gly Ser Leu Asn Thr Leu Val Asp Thr Gln
65 70 75 80
Gly Gln Glu Phe Ile Asp Cys Leu Gly Gly Phe Gly Ile Phe Asn Val
85 90 95
Gly His Arg Asn Pro Val Val Val Ser Ala Val Gln Asn Gln Leu Ala
100 105 110
Lys Gln Pro Leu His Ser Gln Glu Leu Leu Asp Pro Leu Arg Ala Met
115 120 125
Leu Ala Lys Thr Leu Ala Ala Leu Thr Pro Gly Lys Leu Lys Tyr Ser
130 135 140
Phe Phe Cys Asn Ser Gly Thr Glu Ser Val Glu Ala Ala Leu Lys Leu
145 150 155 160
Ala Lys Ala Tyr Gln Ser Pro Arg Gly Lys Phe Thr Phe Ile Ala Thr
165 170 175
Ser Gly Ala Phe His Gly Lys Ser Leu Gly Ala Leu Ser Ala Thr Ala
180 185 190
Lys Ser Thr Phe Arg Lys Pro Phe Met Pro Leu Leu Pro Gly Phe Arg
195 200 205
His Val Pro Phe Gly Asn Ile Glu Ala Met Arg Thr Ala Leu Asn Glu
210 215 220
Cys Lys Lys Thr Gly Asp Asp Val Ala Ala Val Ile Leu Glu Pro Ile
225 230 235 240
Gln Gly Glu Gly Gly Val Ile Leu Pro Pro Pro Gly Tyr Leu Thr Ala
245 250 255
Val Arg Lys Leu Cys Asp Glu Phe Gly Ala Leu Met Ile Leu Asp Glu
260 265 270
Val Gln Thr Gly Met Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His
275 280 285
Glu Asn Val Gln Pro Asp Ile Leu Cys Leu Ala Lys Ala Leu Gly Gly
290 295 300
Gly Val Met Pro Ile Gly Ala Thr Ile Ala Thr Glu Glu Val Phe Ser
305 310 315 320
Val Leu Phe Asp Asn Pro Phe Leu His Thr Thr Thr Phe Gly Gly Asn
325 330 335
Pro Leu Ala Cys Ala Ala Ala Leu Ala Thr Ile Asn Val Leu Leu Glu
340 345 350
Gln Asn Leu Pro Ala Gln Ala Glu Gln Lys Gly Asp Met Leu Leu Asp
355 360 365
Gly Phe Arg Gln Leu Ala Arg Glu Tyr Pro Asp Leu Val Gln Glu Ala
370 375 380
Arg Gly Lys Gly Met Leu Met Ala Ile Glu Phe Val Asp Asn Glu Ile
385 390 395 400
Gly Tyr Asn Phe Ala Ser Glu Met Phe Arg Gln Arg Val Leu Val Ala
405 410 415
Gly Thr Leu Asn Asn Ala Lys Thr Ile Arg Ile Glu Pro Pro Leu Thr
420 425 430
Leu Thr Ile Glu Gln Cys Glu Leu Val Ile Lys Ala Ala Arg Lys Ala
435 440 445
Leu Ala Ala Met Arg Val Ser Val Glu Glu Ala
450 455
<210> 13
<211> 453
<212> PRT
<213>Vibrio fluvialis
<400> 13
Met Asn Lys Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu
1 5 10 15
Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gln Arg Gly Thr Val Val
20 25 30
Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg
35 40 45
Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp
50 55 60
His Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg Phe Pro
65 70 75 80
Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu
85 90 95
Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe
100 105 110
Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu
115 120 125
Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu
130 135 140
Thr Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met
145 150 155 160
Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe
165 170 175
Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu
180 185 190
Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr
195 200 205
Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro
210 215 220
Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr Phe Gln
225 230 235 240
Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile Pro Val Ile Ser Asp
245 250 255
Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val
260 265 270
Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr
275 280 285
Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser
290 295 300
Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly
305 310 315 320
Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala
325 330 335
Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu
340 345 350
Ala Pro Arg Phe Glu Glu Arg Leu Lys His Ile Ala Glu Arg Pro Asn
355 360 365
Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val
370 375 380
Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser
385 390 395 400
Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro
405 410 415
Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala
420 425 430
Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val
435 440 445
Phe Ala Glu Val Ala
450
<210> 14
<211> 224
<212> PRT
<213>Bacillus subtilis
<400> 14
Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu
1 5 10 15
Asn Glu Arg Phe Met Ser Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys
20 25 30
Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu Gly Asp
35 40 45
Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser
50 55 60
Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp
65 70 75 80
Leu Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Val Ile
85 90 95
Cys Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys
100 105 110
Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr
115 120 125
Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr
130 135 140
His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly
145 150 155 160
Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly
165 170 175
Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys
180 185 190
Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His
195 200 205
Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu
210 215 220
<210> 15
<211> 222
<212> PRT
<213>Nocardia species NRRL 5646
<400> 15
Met Ile Glu Thr Ile Leu Pro Ala Gly Val Glu Ser Ala Glu Leu Leu
1 5 10 15
Glu Tyr Pro Glu Asp Leu Lys Ala His Pro Ala Glu Glu His Leu Ile
20 25 30
Ala Lys Ser Val Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala Arg His
35 40 45
Cys Ala Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro Val Ala Ile
50 55 60
Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Val Val Gly
65 70 75 80
Ser Leu Thr His Cys Asp Gly Tyr Arg Ala Ala Ala Val Ala His Lys
85 90 95
Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro His Ala Thr Leu
100 105 110
Pro Glu Gly Val Leu Asp Ser Val Ser Leu Pro Pro Glu Arg Glu Trp
115 120 125
Leu Lys Thr Thr Asp Ser Ala Leu His Leu Asp Arg Leu Leu Phe Cys
130 135 140
Ala Lys Glu Ala Thr Tyr Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp
145 150 155 160
Leu Gly Phe Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser
165 170 175
Ala Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val Pro Gly
180 185 190
Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe Asp Gly Arg Trp
195 200 205
Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala Ile Ala Tyr Ala
210 215 220
<210> 16
<211> 382
<212> PRT
<213>Pseudomonas fluorescens
<400> 16
Met Gln Ile Gln Gly His Tyr Glu Leu Gln Phe Glu Ala Val Arg Glu
1 5 10 15
Ala Phe Ala Ala Leu Phe Asp Asp Pro Gln Glu Arg Gly Ala Gly Leu
20 25 30
Cys Ile Gln Ile Gly Gly Glu Thr Val Val Asp Leu Trp Ala Gly Thr
35 40 45
Ala Asp Lys Asp Gly Thr Glu Ala Trp His Ser Asp Thr Ile Val Asn
50 55 60
Leu Phe Ser Cys Thr Lys Thr Phe Thr Ala Val Thr Ala Leu Gln Leu
65 70 75 80
Val Ala Glu Gly Lys Leu Gln Leu Asp Ala Pro Val Ala Asn Tyr Trp
85 90 95
Pro Glu Phe Ala Ala Ala Gly Lys Glu Ala Ile Thr Leu Arg Gln Leu
100 105 110
Leu Cys His Gln Ala Gly Leu Pro Ala Ile Arg Glu Met Leu Pro Thr
115 120 125
Glu Ala Leu Tyr Asp Trp Arg Leu Met Val Asp Thr Leu Ala Ala Glu
130 135 140
Ala Pro Trp Trp Thr Pro Gly Gln Gly His Gly Tyr Glu Ala Ile Thr
145 150 155 160
Tyr Gly Trp Leu Val Gly Glu Leu Leu Arg Arg Ala Asp Gly Arg Gly
165 170 175
Pro Gly Glu Ser Ile Val Ala Arg Val Ala Arg Pro Leu Gly Leu Asp
180 185 190
Phe His Val Gly Leu Ala Asp Glu Glu Phe Tyr Arg Val Ala His Ile
195 200 205
Ala Arg Ser Lys Gly Asn Met Gly Asp Glu Ala Ala Gln Arg Leu Leu
210 215 220
Gln Val Met Met Arg Glu Pro Thr Ala Met Thr Thr Arg Ala Phe Ala
225 230 235 240
Asn Pro Pro Ser Ile Leu Thr Ser Thr Asn Lys Pro Glu Trp Arg Arg
245 250 255
Met Gln Gln Pro Ala Ala Asn Gly His Gly Asn Ala Arg Ser Leu Ala
260 265 270
Gly Phe Tyr Ser Gly Leu Leu Asp Gly Ser Leu Leu Glu Ala Asp Met
275 280 285
Leu Glu Gln Leu Thr Arg Glu His Ser Ile Gly Pro Asp Lys Thr Leu
290 295 300
Leu Thr Gln Thr Arg Phe Gly Leu Gly Cys Met Leu Asp Gln Gln Pro
305 310 315 320
Gln Leu Pro Asn Ala Thr Phe Gly Leu Gly Pro Arg Ala Phe Gly His
325 330 335
Pro Arg Ser Ala Pro Val Val Arg Trp Val Leu Pro Glu His Asp Val
340 345 350
Ala Phe Gly Phe Val Thr Asn Thr Leu Gly Pro Tyr Val Leu Met Asp
355 360 365
Pro Arg Ala Gln Lys Leu Val Gly Ile Leu Ala Gly Cys Leu
370 375 380
<210> 17
<211> 246
<212> PRT
<213>Lactobacillus brevis
<400> 17
Met Ala Ala Asn Glu Phe Ser Glu Thr His Arg Val Val Tyr Tyr Glu
1 5 10 15
Ala Asp Asp Thr Gly Gln Leu Thr Leu Ala Met Leu Ile Asn Leu Phe
20 25 30
Val Leu Val Ser Glu Asp Gln Asn Asp Ala Leu Gly Leu Ser Thr Ala
35 40 45
Phe Val Gln Ser His Gly Val Gly Trp Val Val Thr Gln Tyr His Leu
50 55 60
His Ile Asp Glu Leu Pro Arg Thr Gly Ala Gln Val Thr Ile Lys Thr
65 70 75 80
Arg Ala Thr Ala Tyr Asn Arg Tyr Phe Ala Tyr Arg Glu Tyr Trp Leu
85 90 95
Leu Asp Asp Ala Gly Gln Val Leu Ala Tyr Gly Glu Gly Ile Trp Val
100 105 110
Thr Met Ser Tyr Ala Thr Arg Lys Ile Thr Thr Ile Pro Ala Glu Val
115 120 125
Met Ala Pro Tyr His Ser Glu Glu Gln Thr Arg Leu Pro Arg Leu Pro
130 135 140
Arg Pro Asp His Phe Asp Glu Ala Val Asn Gln Thr Leu Lys Pro Tyr
145 150 155 160
Thr Val Arg Tyr Phe Asp Ile Asp Gly Asn Gly His Val Asn Asn Ala
165 170 175
His Tyr Phe Asp Trp Met Leu Asp Val Leu Pro Ala Thr Phe Leu Arg
180 185 190
Ala His His Pro Thr Asp Val Lys Ile Arg Phe Glu Asn Glu Val Gln
195 200 205
Tyr Gly His Gln Val Thr Ser Glu Leu Ser Gln Ala Ala Ala Leu Thr
210 215 220
Thr Gln His Met Ile Lys Val Gly Asp Leu Thr Ala Val Lys Ala Thr
225 230 235 240
Ile Gln Trp Asp Asn Arg
245
<210> 18
<211> 261
<212> PRT
<213>Lactobacillus plantarum
<400> 18
Met Ala Thr Leu Gly Ala Asn Ala Ser Leu Tyr Ser Glu Gln His Arg
1 5 10 15
Ile Thr Tyr Tyr Glu Cys Asp Arg Thr Gly Arg Ala Thr Leu Thr Thr
20 25 30
Leu Ile Asp Ile Ala Val Leu Ala Ser Glu Asp Gln Ser Asp Ala Leu
35 40 45
Gly Leu Thr Thr Glu Met Val Gln Ser His Gly Val Gly Trp Val Val
50 55 60
Thr Gln Tyr Ala Ile Asp Ile Thr Arg Met Pro Arg Gln Asp Glu Val
65 70 75 80
Val Thr Ile Ala Val Arg Gly Ser Ala Tyr Asn Pro Tyr Phe Ala Tyr
85 90 95
Arg Glu Phe Trp Ile Arg Asp Ala Asp Gly Gln Gln Leu Ala Tyr Ile
100 105 110
Thr Ser Ile Trp Val Met Met Ser Gln Thr Thr Arg Arg Ile Val Lys
115 120 125
Ile Leu Pro Glu Leu Val Ala Pro Tyr Gln Ser Glu Val Val Lys Arg
130 135 140
Ile Pro Arg Leu Pro Arg Pro Ile Ser Phe Glu Ala Thr Asp Thr Thr
145 150 155 160
Ile Thr Lys Pro Tyr His Val Arg Phe Phe Asp Ile Asp Pro Asn Arg
165 170 175
His Val Asn Asn Ala His Tyr Phe Asp Trp Leu Val Asp Thr Leu Pro
180 185 190
Ala Thr Phe Leu Leu Gln His Asp Leu Val His Val Asp Val Arg Tyr
195 200 205
Glu Asn Glu Val Lys Tyr Gly Gln Thr Val Thr Ala His Ala Asn Ile
210 215 220
Leu Pro Ser Glu Val Ala Asp Gln Val Thr Thr Ser His Leu Ile Glu
225 230 235 240
Val Asp Asp Glu Lys Cys Cys Glu Val Thr Ile Gln Trp Arg Thr Leu
245 250 255
Pro Glu Pro Ile Gln
260
<210> 19
<211> 397
<212> PRT
<213>Treponema denticola
<400> 19
Met Ile Val Lys Pro Met Val Arg Asn Asn Ile Cys Leu Asn Ala His
1 5 10 15
Pro Gln Gly Cys Lys Lys Gly Val Glu Asp Gln Ile Glu Tyr Thr Lys
20 25 30
Lys Arg Ile Thr Ala Glu Val Lys Ala Gly Ala Lys Ala Pro Lys Asn
35 40 45
Val Leu Val Leu Gly Cys Ser Asn Gly Tyr Gly Leu Ala Ser Arg Ile
50 55 60
Thr Ala Ala Phe Gly Tyr Gly Ala Ala Thr Ile Gly Val Ser Phe Glu
65 70 75 80
Lys Ala Gly Ser Glu Thr Lys Tyr Gly Thr Pro Gly Trp Tyr Asn Asn
85 90 95
Leu Ala Phe Asp Glu Ala Ala Lys Arg Glu Gly Leu Tyr Ser Val Thr
100 105 110
Ile Asp Gly Asp Ala Phe Ser Asp Glu Ile Lys Ala Gln Val Ile Glu
115 120 125
Glu Ala Lys Lys Lys Gly Ile Lys Phe Asp Leu Ile Val Tyr Ser Leu
130 135 140
Ala Ser Pro Val Arg Thr Asp Pro Asp Thr Gly Ile Met His Lys Ser
145 150 155 160
Val Leu Lys Pro Phe Gly Lys Thr Phe Thr Gly Lys Thr Val Asp Pro
165 170 175
Phe Thr Gly Glu Leu Lys Glu Ile Ser Ala Glu Pro Ala Asn Asp Glu
180 185 190
Glu Ala Ala Ala Thr Val Lys Val Met Gly Gly Glu Asp Trp Glu Arg
195 200 205
Trp Ile Lys Gln Leu Ser Lys Glu Gly Leu Leu Glu Glu Gly Cys Ile
210 215 220
Thr Leu Ala Tyr Ser Tyr Ile Gly Pro Glu Ala Thr Gln Ala Leu Tyr
225 230 235 240
Arg Lys Gly Thr Ile Gly Lys Ala Lys Glu His Leu Glu Ala Thr Ala
245 250 255
His Arg Leu Asn Lys Glu Asn Pro Ser Ile Arg Ala Phe Val Ser Val
260 265 270
Asn Lys Gly Leu Val Thr Arg Ala Ser Ala Val Ile Pro Val Ile Pro
275 280 285
Leu Tyr Leu Ala Ser Leu Phe Lys Val Met Lys Glu Lys Gly Asn His
290 295 300
Glu Gly Cys Ile Glu Gln Ile Thr Arg Leu Tyr Ala Glu Arg Leu Tyr
305 310 315 320
Arg Lys Asp Gly Thr Ile Pro Val Asp Glu Glu Asn Arg Ile Arg Ile
325 330 335
Asp Asp Trp Glu Leu Glu Glu Asp Val Gln Lys Ala Val Ser Ala Leu
340 345 350
Met Glu Lys Val Thr Gly Glu Asn Ala Glu Ser Leu Thr Asp Leu Ala
355 360 365
Gly Tyr Arg His Asp Phe Leu Ala Ser Asn Gly Phe Asp Val Glu Gly
370 375 380
Ile Asn Tyr Glu Ala Glu Val Glu Arg Phe Asp Arg Ile
385 390 395
<210> 20
<211> 539
<212> PRT
<213>Euglena gracilis
<400> 20
Met Ser Cys Pro Ala Ser Pro Ser Ala Ala Val Val Ser Ala Gly Ala
1 5 10 15
Leu Cys Leu Cys Val Ala Thr Val Leu Leu Ala Thr Gly Ser Asn Pro
20 25 30
Thr Ala Leu Ser Thr Ala Ser Thr Arg Ser Pro Thr Ser Leu Val Arg
35 40 45
Gly Val Asp Arg Gly Leu Met Arg Pro Thr Thr Ala Ala Ala Leu Thr
50 55 60
Thr Met Arg Glu Val Pro Gln Met Ala Glu Gly Phe Ser Gly Glu Ala
65 70 75 80
Thr Ser Ala Trp Ala Ala Ala Gly Pro Gln Trp Ala Ala Pro Leu Val
85 90 95
Ala Ala Ala Ser Ser Ala Leu Ala Leu Trp Trp Trp Ala Ala Arg Arg
100 105 110
Ser Val Arg Arg Pro Leu Ala Ala Leu Ala Glu Leu Pro Thr Ala Val
115 120 125
Thr His Leu Ala Pro Pro Met Ala Met Phe Thr Thr Thr Ala Lys Val
130 135 140
Ile Gln Pro Lys Ile Arg Gly Phe Ile Cys Thr Thr Thr His Pro Ile
145 150 155 160
Gly Cys Glu Lys Arg Val Gln Glu Glu Ile Ala Tyr Ala Arg Ala His
165 170 175
Pro Pro Thr Ser Pro Gly Pro Lys Arg Val Leu Val Ile Gly Cys Ser
180 185 190
Thr Gly Tyr Gly Leu Ser Thr Arg Ile Thr Ala Ala Phe Gly Tyr Gln
195 200 205
Ala Ala Thr Leu Gly Val Phe Leu Ala Gly Pro Pro Thr Lys Gly Arg
210 215 220
Pro Ala Ala Ala Gly Trp Tyr Asn Thr Val Ala Phe Glu Lys Ala Ala
225 230 235 240
Leu Glu Ala Gly Leu Tyr Ala Arg Ser Leu Asn Gly Asp Ala Phe Asp
245 250 255
Ser Thr Thr Lys Ala Arg Thr Val Glu Ala Ile Lys Arg Asp Leu Gly
260 265 270
Thr Val Asp Leu Val Val Tyr Ser Ile Ala Ala Pro Lys Arg Thr Asp
275 280 285
Pro Ala Thr Gly Val Leu His Lys Ala Cys Leu Lys Pro Ile Gly Ala
290 295 300
Thr Tyr Thr Asn Arg Thr Val Asn Thr Asp Lys Ala Glu Val Thr Asp
305 310 315 320
Val Ser Ile Glu Pro Ala Ser Pro Glu Glu Ile Ala Asp Thr Val Lys
325 330 335
Val Met Gly Gly Glu Asp Trp Glu Leu Trp Ile Gln Ala Leu Ser Glu
340 345 350
Ala Gly Val Leu Ala Glu Gly Ala Lys Thr Val Ala Tyr Ser Tyr Ile
355 360 365
Gly Pro Glu Met Thr Trp Pro Val Tyr Trp Ser Gly Thr Ile Gly Glu
370 375 380
Ala Lys Lys Asp Val Glu Lys Ala Ala Lys Arg Ile Thr Gln Gln Tyr
385 390 395 400
Gly Cys Pro Ala Tyr Pro Val Val Ala Lys Ala Leu Val Thr Gln Ala
405 410 415
Ser Ser Ala Ile Pro Val Val Pro Leu Tyr Ile Cys Leu Leu Tyr Arg
420 425 430
Val Met Lys Glu Lys Gly Thr His Glu Gly Cys Ile Glu Gln Met Val
435 440 445
Arg Leu Leu Thr Thr Lys Leu Tyr Pro Glu Asn Gly Ala Pro Ile Val
450 455 460
Asp Glu Ala Gly Arg Val Arg Val Asp Asp Trp Glu Met Ala Glu Asp
465 470 475 480
Val Gln Gln Ala Val Lys Asp Leu Trp Ser Gln Val Ser Thr Ala Asn
485 490 495
Leu Lys Asp Ile Ser Asp Phe Ala Gly Tyr Gln Thr Glu Phe Leu Arg
500 505 510
Leu Phe Gly Phe Gly Ile Asp Gly Val Asp Tyr Asp Gln Pro Val Asp
515 520 525
Val Glu Ala Asp Leu Pro Ser Ala Ala Gln Gln
530 535
<210> 21
<211> 269
<212> PRT
<213>Bacillus cercus
<400> 21
Met Ile Asn Lys Thr Leu Leu Gln Lys Arg Phe Asn Gly Ala Ala Val
1 5 10 15
Ser Tyr Asp Arg Tyr Ala Asn Val Gln Lys Lys Met Ala His Ser Leu
20 25 30
Leu Ser Ile Leu Lys Glu Arg Tyr Ser Glu Thr Ala Ser Ile Arg Ile
35 40 45
Leu Glu Leu Gly Cys Gly Thr Gly Tyr Val Thr Glu Gln Leu Ser Lys
50 55 60
Leu Phe Pro Lys Ser His Ile Thr Ala Val Asp Phe Ala Glu Ser Met
65 70 75 80
Ile Ala Ile Ala Gln Thr Arg Gln Asn Val Lys Asn Val Thr Phe His
85 90 95
Cys Glu Asp Ile Glu Arg Leu Arg Leu Glu Glu Ser Tyr Asp Val Ile
100 105 110
Ile Ser Asn Ala Thr Phe Gln Trp Leu Asn Asn Leu Gln Gln Val Leu
115 120 125
Arg Asn Leu Phe Gln His Leu Ser Ile Asp Gly Ile Leu Leu Phe Ser
130 135 140
Thr Phe Gly His Glu Thr Phe Gln Glu Leu His Ala Ser Phe Gln Arg
145 150 155 160
Ala Lys Glu Glu Arg Asn Ile Lys Asn Glu Thr Ser Ile Gly Gln Arg
165 170 175
Phe Tyr Ser Lys Asp Gln Leu Leu His Ile Cys Lys Ile Glu Thr Gly
180 185 190
Asp Val His Val Ser Glu Thr Cys Tyr Ile Glu Ser Phe Thr Glu Val
195 200 205
Lys Glu Phe Leu His Ser Ile Arg Lys Val Gly Ala Thr Asn Ser Asn
210 215 220
Glu Gly Ser Tyr Cys Gln Ser Pro Ser Leu Phe Arg Ala Met Leu Arg
225 230 235 240
Ile Tyr Glu Arg Asp Phe Thr Gly Asn Glu Gly Ile Met Ala Thr Tyr
245 250 255
His Ala Leu Phe Ile His Ile Thr Lys Glu Gly Lys Arg
260 265
<210> 22
<211> 132
<212> PRT
<213>Escherichia coli
<400> 22
Met Ser Thr Thr His Asn Val Pro Gln Gly Asp Leu Val Leu Arg Thr
1 5 10 15
Leu Ala Met Pro Ala Asp Thr Asn Ala Asn Gly Asp Ile Phe Gly Gly
20 25 30
Trp Leu Met Ser Gln Met Asp Ile Gly Gly Ala Ile Leu Ala Lys Glu
35 40 45
Ile Ala His Gly Arg Val Val Thr Val Arg Val Glu Gly Met Thr Phe
50 55 60
Leu Arg Pro Val Ala Val Gly Asp Val Val Cys Cys Tyr Ala Arg Cys
65 70 75 80
Val Gln Lys Gly Thr Thr Ser Val Ser Ile Asn Ile Glu Val Trp Val
85 90 95
Lys Lys Val Ala Ser Glu Pro Ile Gly Gln Arg Tyr Lys Ala Thr Glu
100 105 110
Ala Leu Phe Lys Tyr Val Ala Val Asp Pro Glu Gly Lys Pro Arg Ala
115 120 125
Leu Pro Val Glu
130
<210> 23
<211> 286
<212> PRT
<213>Escherichia coli
<400> 23
Met Ser Gln Ala Leu Lys Asn Leu Leu Thr Leu Leu Asn Leu Glu Lys
1 5 10 15
Ile Glu Glu Gly Leu Phe Arg Gly Gln Ser Glu Asp Leu Gly Leu Arg
20 25 30
Gln Val Phe Gly Gly Gln Val Val Gly Gln Ala Leu Tyr Ala Ala Lys
35 40 45
Glu Thr Val Pro Glu Glu Arg Leu Val His Ser Phe His Ser Tyr Phe
50 55 60
Leu Arg Pro Gly Asp Ser Lys Lys Pro Ile Ile Tyr Asp Val Glu Thr
65 70 75 80
Leu Arg Asp Gly Asn Ser Phe Ser Ala Arg Arg Val Ala Ala Ile Gln
85 90 95
Asn Gly Lys Pro Ile Phe Tyr Met Thr Ala Ser Phe Gln Ala Pro Glu
100 105 110
Ala Gly Phe Glu His Gln Lys Thr Met Pro Ser Ala Pro Ala Pro Asp
115 120 125
Gly Leu Pro Ser Glu Thr Gln Ile Ala Gln Ser Leu Ala His Leu Leu
130 135 140
Pro Pro Val Leu Lys Asp Lys Phe Ile Cys Asp Arg Pro Leu Glu Val
145 150 155 160
Arg Pro Val Glu Phe His Asn Pro Leu Lys Gly His Val Ala Glu Pro
165 170 175
His Arg Gln Val Trp Ile Arg Ala Asn Gly Ser Val Pro Asp Asp Leu
180 185 190
Arg Val His Gln Tyr Leu Leu Gly Tyr Ala Ser Asp Leu Asn Phe Leu
195 200 205
Pro Val Ala Leu Gln Pro His Gly Ile Gly Phe Leu Glu Pro Gly Ile
210 215 220
Gln Ile Ala Thr Ile Asp His Ser Met Trp Phe His Arg Pro Phe Asn
225 230 235 240
Leu Asn Glu Trp Leu Leu Tyr Ser Val Glu Ser Thr Ser Ala Ser Ser
245 250 255
Ala Arg Gly Phe Val Arg Gly Glu Phe Tyr Thr Gln Asp Gly Val Leu
260 265 270
Val Ala Ser Thr Val Gln Glu Gly Val Met Arg Asn His Asn
275 280 285

Claims (71)

1. in recombinant host biosynthesis glutaric acid methyl esters method, methods described includes thering is malonyl using at least one The polypeptide and at least one polypeptide with thioesterase activity of base-CoA O- methyl transferase activities are in the host by the third two At least one enzymatic in acyl group-[acp] and malonyl-CoA is converted into glutaric acid methyl esters.
2. the method for claim 1 wherein using described at least one with malonyl-CoA O- methyl transferase activities Malonyl-[acp] enzymatic is converted into malonyl-[acp] methyl esters by polypeptide.
3. the method for claim 2, wherein using it is at least one with the active polypeptide being selected from the group by malonyl- [acp] methyl esters enzymatic is converted into glutaryl-[acp] methyl esters：Synthase activity, dehydrogenase activity, Dehydratase activity, and reductase Activity.
4. the method for claim 3, wherein using at least one polypeptide with thioesterase activity by glutaryl- [acp] methyl esters enzymatic is converted into glutaric acid methyl esters.
5. the method for claim 1 wherein using described at least one with malonyl-CoA O- methyl transferase activities Malonyl-CoA enzymatics are converted into malonyl-CoA methyl esters by polypeptide.
6. the method for claim 5, wherein using it is at least one with the active polypeptide being selected from the group by malonyl-CoA Methyl esters enzymatic is converted into glutaryl-CoA methyl esters：Synthase activity, beta-Ketothiolase activity, dehydrogenase activity, hydratase activity, And reductase activity.
7. the method for claim 6, wherein using at least one polypeptide with thioesterase activity by glutaryl-CoA Methyl esters enzymatic is converted into glutaric acid methyl esters.
8. the method for any one of claim 1-7, wherein described with many of malonyl-CoA O- methyl transferase activities Peptide and SEQ ID NO:The amino acid sequence listed in 21 has at least 70% sequence identity.
9. the method for any one of claim 3,4 or 6-8, wherein the polypeptide with reductase activity and SEQ ID NO:The amino acid sequence listed in 19 or 20 has at least 70% sequence identity.
10. the method for any one of claim 1-9, methods described further includes there is carboxylate reductase using at least one Glutaric acid methyl esters enzymatic is converted into glutaric acid semialdehyde methyl esters by the polypeptide of activity in the host.
The method of 11. claims 10, its further include using it is at least one with the active polypeptide being selected from the group by penta Diacid semialdehyde methyl esters enzymatic is converted into 5- aminovaleric acids：ω-transaminase activity and esterase active.
The method of 12. claims 11, its further include using it is at least one with the active polypeptide being selected from the group by 5- Aminovaleric acid enzymatic is converted into cadaverine：Carboxylate reductase activity, ω-transaminase activity, N- acetyl transferase activities, alcohol dehydrogenase Enzymatic activity, and deacetylase activity
The method of 13. claims 1, methods described is further included using at least one with the active polypeptide being selected from the group Glutaric acid methyl esters enzymatic is converted into 5- oxopentanoic acids：Carboxylate reductase activity and esterase active.
The method of 14. claims 13, methods described is further included using at least one active many with what is be selected from the group 5- oxopentanoic acid enzymatics are converted into cadaverine by peptide：Carboxylate reductase activity, and ω-transaminase activity activity.
The method of 15. claims 10, methods described further includes to use at least one polypeptide with esterase active by penta Diacid semialdehyde methyl esters enzymatic is converted into 5- hydroxypentanoic acids.
The method of 16. claims 15, wherein the polypeptide with esterase active and SEQ ID NO:The amino listed in 16 Acid sequence has at least 70% sequence identity.
The method of 17. claims 15 or claim 16, it further includes to use at least one with dehydrogenase activity Glutaric acid semialdehyde methyl esters enzymatic is converted into 5- hydroxypentanoic acids by polypeptide.
The method of any one of 18. claim 15-17, it is further included using at least one with the activity being selected from the group Polypeptide 5- hydroxypentanoic acid enzymatics are converted into cadaverine：Carboxylate reductase activity, ω-transaminase activity, and alcohol dehydrogenase activity.
The method of any one of 19. claim 15-17, it is further included using at least one with the activity being selected from the group Polypeptide 5- hydroxypentanoic acid enzymatics are converted into 1,5- pentanediols：Carboxylate reductase activity and alcohol dehydrogenase activity.
The method of 20. claims 19, its further include using it is at least one with the active polypeptide being selected from the group by 1, 5- pentanediol enzymatics are converted into cadaverine：ω-transaminase activity and alcohol dehydrogenase activity.
The method of any one of 21. claim 1-9, methods described further includes to use at least one with esterase active Glutaric acid methyl esters enzymatic is converted into glutaric acid by polypeptide.
The method of 22. claims 21, methods described is further included using at least one with many of carboxylate reductase activity Glutaric acid enzymatic is converted into 5- aminovaleric acids by peptide and at least one polypeptide with ω-transaminase activity.
The method of 23. claims 21, methods described is further included using at least one with many of carboxylate reductase activity Glutaric acid enzymatic is converted into 5- hydroxyls penta by the polypeptide of the dehydrogenase activity classified under peptide and at least one 1.1.1.- with EC Acid.
The method of any one of 24. claim 10-20,22 and 23, wherein it is described with carboxylate reductase activity polypeptide with SEQ ID NO:The amino acid sequence of any one listed in 2-7 has at least 70% sequence identity.
The method of any one of 25. claim 1-22, wherein the polypeptide with thioesterase activity and SEQ ID NO:17, 18, any one amino acid sequence listed in 22, and 23 has at least 70% sequence identity.
26. claims 11, any one of 12,14,18,20, and 22 method, wherein described with ω-transaminase activity Polypeptide and SEQ ID NO:Any one amino acid sequence listed in 8-13 has at least 70% sequence identity.
The method of 27. generation glutaric acids, methods described includes that (i) is used with heptanedioyl-[acp] methyl ester methyl esterase active Glutaryl-[acp] methyl esters enzymatic is converted into glutaryl-[acp] or converts glutaryl-CoA methyl esters enzymatic by polypeptide It is glutaryl-CoA, and (ii) has thioesterase activity, reversible CoA ligase activity, CoA transfers using at least one Enzymatic activity, acylated dehydrogenase activity, aldehyde dehydrogenase activity, glutarate-semialdehyde dehydrogenase activity, or succinic semialdehyde dehydrogenase is lived Glutaryl-[acp] or glutaryl-CoA enzymatics are converted into glutaric acid by the polypeptide of property.
The method of 28. claims 27, wherein it is described with heptanedioyl-polypeptide and SEQ of [acp] methyl ester methyl esterase active ID NO:The amino acid sequence listed in 1 has at least 70% sequence identity.
The method of 29. claims 27 or claim 28, wherein using with thioesterase activity polypeptide by glutaryl- [acp] or glutaryl-CoA enzymatics are converted into glutaric acid.
The method of 30. claims 27 or claim 28, wherein being shifted using with reversible CoA ligase activity or CoA Glutaryl-[acp] or glutaryl-CoA enzymatics are converted into glutaric acid by the polypeptide of enzymatic activity.
The method of 31. claims 27 or claim 28, wherein dehydrogenase activity is acylated using having, aldehyde dehydrogenase activity, Glutarate-semialdehyde dehydrogenase activity, or succinic semialdehyde dehydrogenase activity polypeptide by glutaryl-[acp] or glutaryl- CoA enzymatics are converted into glutaric acid.
The method of any one of 32. claim 1-31, wherein the host undergoes the training under aerobic or micro- aerobic condition of culture Support strategy.
The method of any one of 33. claim 1-32, wherein being limited under conditions of nutrition limitation via nitrogen, phosphate or oxygen Cultivate the host.
The method of any one of 34. claim 1-33, wherein retaining the host to maintain during fermentation using ceramic membrane High-cell density.
The method of any one of 35. claim 1-34, wherein feed supplement are derived from biological raw material to the primary carbon source of fermentation.
The method of 36. claims 35, wherein the biological raw material is derived from monose, disaccharides, lignocellulosic, hemicellulose, Cellulose, lignin, levulic acid and formic acid, triglycerides, glycerine, aliphatic acid, agricultural wastes, concentration vinasse (condensed ) or municipal waste distillers'solubles.
The method of any one of 37. claim 1-34, wherein feed supplement are derived from abiotic raw material to the primary carbon source of fermentation.
The method of 38. claims 37, wherein the abiotic raw material is derived from natural gas, synthesis gas, CO2/H2, methyl alcohol, second Alcohol, benzoate, non-volatile residue (NVR) or alkali wash water (caustic wash) waste from cyclohexane oxidation process Stream or terephthalic acid (TPA)/isophathalic acid mixture waste stream.
The method of any one of 39. claim 1-38, wherein the host is prokaryotes.
The method of 40. claims 39, wherein the prokaryotes are selected from the group：Escherichia (Escherichia)；Clostridium Category (Clostridia)；Corynebacterium (Corynebacteria)；Greedy copper Pseudomonas (Cupriavidus)；Pseudomonas (Pseudomonas)；Delftiatsuruhatensis belongs to (Delftia)；Bacillus (Bacilluss)；Lactobacillus (Lactobacillus)；Lactococcus (Lactococcus)；With Rhod (Rhodococcus).
The method of 41. claims 40, wherein the prokaryotes are selected from the group：Escherichia coli (Escherichia coli), Young clostridium (Clostridium ljungdahlii), from producing and ethanol clostridium (Clostridium autoethanogenum), Clostridium kluyveri (Clostridium kluyveri), corynebacterium glutamicum (Corynebacterium glutamicum), Hookworm corrupt bacteria (Cupriavidus necator), resistance to metal covet copper bacterium (Cupriavidus metallidurans), fluorescence Pseudomonad (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida), edible oil vacation unit cell Bacterium (Pseudomonas oleavorans), acidophilic bacteria (Delftia acidovorans), Bacillus subtillis (Bacillus subtillis), Lactobacillus delbrueckii (Lactobacillus delbrueckii), Lactococcus lactis (Lactococcus lactis) and Rhodococcus equi (Rhodococcus equi).
The method of any one of 42. claim 1-38, wherein the host is eucaryote.
The method of 43. claims 42, wherein the eucaryote is selected from the group：Aspergillus (Aspergillus), saccharomyces (Saccharomyces) Chi Shi ferment category (Pichia), Ye Luoweiya saccharomyces (Yarrowia), Issatchenkia, are finished (Issatchenkia), Debaryomyces (Debaryomyces), Arxula and Kluyveromyces (Kluyveromyces)。
The method of 44. claims 43, wherein the eucaryote is selected from the group：Aspergillus niger (Aspergillus niger), wine Brewer yeast (Saccharomyces cerevisiae), pichia pastoris phaff (Pichia pastoris), solution fat Ye Luoweiya Yeast (Yarrowia lipolytica), Issatchenkia orientalis (Issathenkia orientalis), the inferior Dbaly yeast of the Chinese (Debaryomyces hansenii), Arxula adenoinivorans and lactic acid yeast kluyveromyces (Kluyveromyces lactis)。
The method of any one of 45. claim 1-44, wherein the host shows the tolerance to high concentration C5 building blocks, And wherein improve the tolerance to high concentration C5 building blocks via the continuous culture in selective environment.
The method of any one of 46. claim 1-45, wherein the allogenic polypeptide below one or more of host expresses, institute Stating allogenic polypeptide has acetyl-CoA synzyme, 6-phosphogluconate dehydrogenase；Transketolase；Feed back resistance threonine takes off Ammonia enzyme；Pyridine nucleotide transhydrogenase；Hydrogenlyase；Glyceraldehyde -3P- dehydrogenases；Malate dehydrogenase；G-6-P dehydrogenation Enzyme；The diphosphatase of fructose 1,6；Propiono-CoA synzyme；L-alanine dehydrogenase；Pidolidone dehydrogenase；Glu Synzyme；Lysine transport protein；Dicarboxylic acids transport protein；And/or multidrug transporter activity.
The method of any one of 47. claim 1-46, wherein the host comprising have active one kind for being selected from the group or The decrease of multiple polypeptides：The specific beta-Ketothiolase of polyhydroxyalkanoate synthase, acetyl-CoA thioesterase, acetyl-CoA, Acetoacetyl-CoA reductases, the phosphoric acid acetic acid transferase, acetokinase, lactic dehydrogenase, the menaquinol- that form acetic acid Fumaric acid oxidoreducing enzyme, 2- ketone acids (oxoacid) decarboxylase for producing isobutanol, alcohol dehydrogenase, the triose for forming ethanol Phosphoric acid isomerase, pyruvate decarboxylase, GPI, the unbalanced transhydrogenase of dissipation co-factor, to create not The specific glutamte dehydrogenase of co-factor of balance, the glutamte dehydrogenase using NADH/NADPH, heptanedioyl-CoA dehydrogenations Enzyme；Receive the acyl-CoA dehydrogenase of C5 building blocks and center precursor as substrate；Glutaryl-CoA dehydrogenases；With heptan two Acyl-CoA synzyme.
48. recombinant host cells, it includes at least one polypeptide of the coding with malonyl-CoA O- methyl transferase activities With the exogenous nucleic acid of the polypeptide with thioesterase activity, host's generation glutaric acid methyl esters.
The host of 49. claims 42, the host is further comprising the allogenic polypeptide with carboxylate reductase activity, the place Main further generation glutaric acid semialdehyde methyl esters.
The host of 50. claims 48 or claim 49, the host further comprising one or more have be selected from the group Active allogenic polypeptide：Synthase activity, dehydrogenase activity, Dehydratase activity, and reductase activity.
The host of 51. claims 48 or claim 49, the host further comprising one or more have be selected from the group Active allogenic polypeptide：Synthase activity, beta-Ketothiolase activity, dehydrogenase activity, hydratase activity, and reductase activity.
The host of any one of 52. claim 48-51, wherein described with malonyl-CoA O- methyl transferase activities Polypeptide and SEQ ID NO:The amino acid sequence listed in 21 has at least 70% sequence identity.
The host of any one of 53. claim 48-52, wherein the polypeptide with thioesterase activity and SEQ ID NO: The amino acid sequence that any one of 17-18 is listed has at least 70% sequence identity.
The host of any one of 54. claims 50 and 51, wherein the polypeptide with reductase activity and SEQ ID NO: Any one amino acid sequence listed in 19 or 20 has at least 70% sequence identity.
The host of any one of 55. claim 48-53, the host further includes the allogenic polypeptide with esterase active, The host further generates glutaric acid or 5- oxopentanoic acids.
The host of 56. claims 55, the host is also comprising the allogenic polypeptide with ω-transaminase activity, host's life Into 5- aminovaleric acids.
The host of any one of 57. claims 4 and 50-54, the host also has what is be selected from the group comprising one or more The allogenic polypeptide of activity：ω-transaminase activity, carboxylate reductase activity and esterase active, the host generate 5- aminovaleric acids.
The host of 58. claims 56 or claim 57, the host has the activity being selected from the group comprising one or more Allogenic polypeptide：Carboxylate reductase activity, N- acetyl transferase activities and deacetylase activity, the host is from 5- amino Valeric acid generates cadaverine.
The host of 59. claims 55, the host also has the active allogenic polypeptide being selected from the group comprising one or more： Carboxylate reductase activity and ω-transaminase activity, the host generate cadaverine from 5- oxopentanoic acids.
The host of any one of 60. claim 48-54, the host is active with what is be selected from the group comprising one or more Allogenic polypeptide：Esterase active, 6 hydroxycaproic acid dehydrogenase activity, 4 hydroxybutyric acid dehydrogenase activity, 5- hydroxypentanoic acid dehydrogenases Activity, and alcohol dehydrogenase activity, the host generate 5- hydroxypentanoic acids.
The host of 61. claims 60, the host has the active allogenic polypeptide being selected from the group comprising one or more：Carboxylic Sour reductase activity and alcohol dehydrogenase activity, the host generate 1,5-PD from 5- hydroxypentanoic acids.
The host of 62. claims 61, the host has the active allogenic polypeptide being selected from the group comprising one or more：Alcohol Dehydrogenase activity and ω-transaminase activity, the host produce cadaverine from 1,5-PD.
The host of 63. claims 60, the host has the active allogenic polypeptide being selected from the group comprising one or more：Carboxylic Sour reductase activity, alcohol dehydrogenase activity and ω-transaminase activity, the host generate cadaverine from 5- hydroxypentanoic acids.
The host of any one of 64. claim 49-53 and 55-63, wherein it is described with carboxylate reductase activity polypeptide with SEQ ID NO:Any one amino acid sequence listed in any one of 2-7 has at least 70% sequence identity.
The host of any one of 65. claim 57-59 and 62-64, wherein the polypeptide with ω-transaminase activity with SEQ ID NO:Any one amino acid sequence listed in 8-13 has at least 70% sequence identity.
66. recombinant hosts, it includes at least one exogenous nucleic acid, and the exogenous nucleic acid coding has heptanedioyl-[acp] methyl esters first The polypeptide of base ester enzymatic activity, and it is at least one with the active polypeptide being selected from the group：Thioesterase activity, reversible CoA connections Enzymatic activity, CoA transferase actives, acylated dehydrogenase activity, aldehyde dehydrogenase activity, glutarate-semialdehyde dehydrogenase activity, and amber Sour semialdehyde dehydrogenase activity.
The recombinant host of 67. claims 66, wherein it is described with heptanedioyl-polypeptide of [acp] methyl ester methyl esterase active with SEQ ID NO:The amino acid sequence listed in 1 has at least 70% sequence identity.
68. biologically-derived products, the product based on biology or the derivative product of fermentation, wherein the product is included：
I. composition, the composition is included according to any one of claim 1-42, or any one of Fig. 1-9 at least one Biologically-derived, based on biology or compound derived from fermentation, or its any combinations,
Ii. it is biologically-derived, based on the biological or derivative polymer of fermentation, its include i. it is biologically-derived, based on biology Or the derivative composition of fermentation or compound, or its any combinations,
Iii. the derivative resin of biologically-derived, based on biology or fermentation, it includes the biologically-derived, based on biology of i. Or the derivative compound of fermentation or the derivative composition of biologically-derived, based on biology or fermentation or its any combinations or ii. It is biologically-derived, based on biological or the derivative polymer of fermentation or its any combinations,
Iv. molding substance, by making, ii.'s is biologically-derived, based on the biological or derivative polymer of fermentation or iii. for it Biologically-derived, based on biology or ferment derivative resin or the molding acquisition of its any combinations,
V. the derivative preparaton of biologically-derived, based on biology or fermentation, it includes the biologically-derived, based on biology of i. Or the derivative composition of fermentation, i. it is biologically-derived, based on the biological or derivative compound of fermentation, ii. it is biologically-derived , based on biological or the derivative polymer of fermentation, iii. it is biologically-derived, based on the biological or derivative resin of fermentation, or The derivative molding substance of biologically-derived, based on biology or fermentation of iv, or its any combinations, or
Vi. the derivative semi-solid or non-semisolid flow of biologically-derived, based on biology or fermentation, it includes the biologically-derived of i. , based on the biological or derivative composition of fermentation, i. it is biologically-derived, based on the biological or derivative compound of fermentation, Ii. it is biologically-derived, based on biological or the derivative polymer of fermentation, iii. it is biologically-derived, based on biological or fermentation Derivative resin, v. is biologically-derived, based on the biological or derivative preparaton of fermentation, or iv. it is biologically-derived, based on life Thing or the derivative molding substance of fermentation, or its any combinations.
69. improve the polypeptide with carboxylate reductase activity to substituted or unsubstituted C4-C8The active method of dicarboxylic acids, Methods described includes using the polypeptide with malonyl-CoA methyl transferase activities by the C4-C8Dicarboxylic acids enzymatic is converted It is HOC (=O) (C2-C6Alkyl)-C (=O) OCH3Ester, uses the polypeptide with carboxylate reductase activity by HOC (=O) afterwards (C2-C6Alkyl)-C (=O) OCH3Esterase promotees to be converted into HC (=O) (C2-C6Alkyl)-C (=O) OCH3。
The method of 70. claims 69, it further includes to use the polypeptide with dehydrogenase activity by the HC (=O) (C2- C6Alkyl)-C (=O) OCH3It is converted into HOCH2(C2-C6 alkyl)-C (=O) OCH3。
The method of 71. claims 70, it further includes to use the polypeptide with esterase active by HOCH2(C2-C6Alkyl)-C (=O) OCH3Enzymatic is converted into HOCH2(C2-C6Alkyl (alkyl))-C (=O) OH.
CN201580038453.6A 2014-06-16 2015-06-16 Method for generating glutaric acid and glutaric acid methyl esters Pending CN106795519A (en)

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